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Use of topically applied cyclosporine to enhance corneal sensitivity restoration rate in an eye of an individual after ocular surgery such as laser-assisted in situ keratomileusis (LASIK) in which nerves are severed.
1. A method of enhancing the rate of restoration of corneal sensitivity in an eye of an individual after refractive surgery, the method comprising
topically administering cyclosporine to an eye of an individual having loss of corneal sensation after refractive surgery on the eye under conditions of at least daily dosing intervals in a therapeutic regimen for a duration sufficient to provide an enhanced rate of restoration of corneal sensitivity in the individual after refractive surgery, and assessing the enhanced rate of restoration of post-surgical corneal sensitivity.
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topically administering cyclosporine to an eye of an individual having loss of corneal sensation after refractive surgery on the eye,
and assessing the enhanced rate of restoration of post-surgical corneal sensitivity.
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This application is a Continuation-In-Part of U.S. patent application Ser. No. 11/263,737 filed Nov. 1, 2005, which is a Continuation-In-Part of U.S. patent application Ser. No. 11/183,355 filed Jul. 18, 2005, each of which is expressly incorporated by reference herein in its entirety.
Methods to enhance corneal sensation and/or reduce scarring after ocular surgery, and to enhance ocular neuroprotection and/or neurostimulation to reduce neurodegenerative changes that may be associated with glaucoma or other ocular diseases.
Methods and compositions that enhance a patient's condition prophylactically or therapeutically, or after ocular surgery are desirable.
One embodiment is a composition comprising at least one neuro-stimulatory factor in a pharmaceutically effective concentration and formulation for non-systemic localized ocular administration and effect. The composition contains macrolides or may further be modified to contain one or more macrolides if not already present. It may be formulated with excipients for topical ocular administration, administration in an ocular device or delayed release matrix, administration by subconjunctival or intraocular injection, etc. It may be contained in an intraocular implant, an intraocular lens, or a contact lens. The neuroprotective or neurostimulatory factor may be a macrolide, which may be cyclosporine, tacrolimus, sirolimus, everolimus, pimocrolous, or others; macrolide analog; neurotrophin; and/or a neuropoietic factor. In some embodiments, one or more other agents may also be included, for example, an antioxidant, steroid, non-steroidal anti-inflammatory drug, antibiotic, anti-proliferative agent, anti-cell migration agent, anti-prostaglandin, anti-angiogenic agent, vitamin, mineral, growth factor, or cytokine.
Another embodiment is an ocular method comprising administering to a patient after ocular surgery a composition comprising at least one neurostimulatory factor, which also encompasses a macrolide or macrolide analog with neurostimulatory activity, in a pharmaceutically effective concentration and formulation for non-systemic localized ocular administration. The composition may be ocularly administered topically, subconjunctivally, intraocularly, by implantation in a device or a lens, or from a contact lens. The composition may be administered to the patient after corneal surgeries or procedures such as laser-assisted in situ keratomileusis (LASIK), laser-assisted in situ epithelial keratomileusis (LASEK), photorefractive keratectomy (PRK), keratoplasty (total corneal transplant, partial corneal transplant), etc.
Another embodiment is an ocular method whereby a macrolide or macrolide analog is administered to a post-ocular surgery patient to reduce or minimize ocular scarring. The macrolide may be present as a component in a composition administered to provide a neuroprotective and/or neurostimulatory effect. Alternatively, the macrolide may be administered to reduce or minimize scarring following any type of ocular surgery, including but not limited to glaucoma surgery, retinal detachment repair surgery, and corneal surgery.
Another embodiment is a method for administering a macrolide, macrolide analog, neurotrophin, and/or neuropoietic agent prophylactically to patients having or at risk for developing an ocular neurologic or neurosensory disease, or therapeutically to patients with an ocular neurologic or neurosensory disease, either alone or in conjunction with other therapy. Glaucoma is a non-limiting example of an ocular disease with a neuroassociated component. Retinitis pigmentosa is a non-limiting example of an ocular disease with a neurosensory component.
Another embodiment discloses topical ocular cyclosporine to enhance recovery of corneal sensitivity in patients after corneal incisional or lamellar surgeries or procedures such as LASIK, LASEK, PRK, etc. Topical ocular cyclosporine applied post-LASIK surgery has reduced inflammatory processes and enhanced secretion of a tear film component, thereby reducing symptoms of dry eye; however, topical ocular cyclosporine efficacy in enhancement of post-LASIK corneal sensitivity recovery rate has not previously been demonstrated prior to the present invention. Topical ocular cyclosporine effect on enhancement in ocular nerve regeneration has not previously been suggested prior to the present invention.
These and other embodiments of the invention will be further appreciated in view of the following drawings, detailed description, and examples.
One embodiment is a method to enhance patient recovery after ocular surgery or other trauma by enhancing corneal sensation, ocular nerve regeneration, and/or re-enervation. The method enhances the restoration rate and restores at least partially the loss of corneal sensation that occurs following corneal procedures during which nerves are severed. The method also reduces or minimizes post-surgical scarring that could lead to corneal opacification, reduced vision, and/or other complications, by compositions with a macrolide or macrolide analog component. For example, it could be used to reduce or minimize scarring of the conjunctiva that occurs after glaucoma surgery, or scarring that may lead to proliferative vitreal retinopathy (PVR) after retinal detachment repair surgery, or scarring that occurs after corneal surgery. While not being bound by a specific theory, a method to reduce or minimize post ocular-surgery scarring may enhance ocular sensation, nerve regeneration, and/or re-enervation, possibly by minimizing scar tissue that may impair nerve growth, nerve cell connections, etc. The method thus leads to enhanced recovery following ocular surgery.
One embodiment is a method to enhance ocular neuroprotection and/or neurostimulation in or to an eye by locally administering to the eye, in a non-systemic manner, a composition of at least one neuroprotective or neurostimulatory factor. The neuroprotective or neurostimulatory factor may be administered either alone or in combination with other agents such as neurotrophins, neuropoietins, etc. The neurostimulatory and/or neuroprotective factor may be, but is not limited to, a macrolide, a macrolide analog, a neurotrophin, and/or a neuropoietic agent.
One embodiment provides localized ocular administration of macrolides and/or macrolide analogs, either alone or in combination with other neuro-stimulatory agents such as neurotrophins, neuropoietins, etc. The macrolides and/or macrolide analogs may or may not have neuro-stimulatory activity.
"Corneal anesthesia" is an unwanted consequence in some patients who have undergone an ocular surgical procedure. Such procedures include laser-assisted in situ keratomileusis (LASIK), laser-assisted in situ epithelial keratomileusis (LASEK), photorefractive keratectomy (PRK), and keratoplasty, i.e., corneal transplant (total or partial). In some of these types of procedures, the surgeon creates a micro-thin flap (shown in
The stromal corneal flap may be created using a femtosecond computer-guided laser, or a hand-held microkeratome with an oscillating metal blade. The flap is then folded open to provide access to the cornea for the procedure, after which the flap is then closed by returning to its original position. It seals without stitches and heals shut. The flap promotes post-surgical healing, patient comfort, and improved vision. If the flap is not of the proper thickness (e.g., too thick, too thin, or irregular), the patient's healing and quality of vision may be compromised.
In creating the LASIK flap, the nerves that enervate the surface of the cornea are mechanically dissected. This results in corneal nerve destruction and, consequently, reduced corneal sensitivity within the LASIK flap area. One study reported that the number of sub-basal and stromal nerve fiber bundles in the corneal flap decreased 90% immediately following surgery. Although the sub-basal nerve fiber bundles gradually returned, their number remained less than half of the pre-surgical number. The loss of corneal sensation caused by a decrease in the number of enervating nerves, and/or their function, may last up to about six months after the original procedure. Diabetic patients are particularly prone to decreased corneal nerve function, yet are a group of patients in frequent need of corneal transplants.
After surgery corneal nerve fibers gradually re-innervate the cornea, and corneal sensation is slowly restored. Complete corneal recovery after LASIK is a very slow process, and compromised corneal sensitivity is a precarious situation. A low corneal sensitivity threshold is required for early detection of foreign bodies, injury, and/or pathological changes. The loss of normal corneal sensation can compromise the protective blink reflex, delay epithelial wound healing, decrease tear flow, and be associated with neurotrophic keratilis, sterile corneal melts, and/or infectious keratides. At least six months is necessary for full restoration of corneal sensitivity, even longer for patients with higher degrees of myopia. Complete nerve restoration (i.e., re-enervation) to pre-surgical density levels is not complete before five years post-LASIK.
In general, LASIK, LASEK, PRK, and other types of corneal incisional or lamellar surgeries or procedures are performed on an outpatient basis. Prior to surgery, a topical anesthesia is applied to the eye, and a speculum is placed to separate the eyelids.
LASEK is similar to LASIK but in LASEK a corneal epithelial flap is cut and ablation is performed on the surface, followed by repositioning of the epithelial flap.
In PRK, the surface corneal epithelia are removed. Laser ablation to reshape the corneal surface commences, with pre-programmed parameters based upon specific patient vision characteristics (e.g., myopia, hyperopia, astigmatism, etc.).
Laser thermal keratoplasty (LTK) uses a holmium-YAG laser energy to reshape the cornea. Laser energy is applied in a predetermined pattern to shrink collagen fibers and thus steepen the cornea. Ocular tissue is neither cut nor removed.
Conductive keratoplasty (CK) is a non-laser based ocular procedure. It does not involve cutting the cornea, and reshapes the cornea using low-level radiofrequency (RF) energy administered through a fine probe. Using the probe, RF energy is applied to specific sites in a pre-set marked circular pattern (e.g., using a dye) on the outer cornea to shrink or ablate small areas of corneal tissue. The circular shrinkage pattern creates a constrictive band, thus increasing the overall curvature of the cornea.
In keratoplasty or penetrating keratoplasty, the cornea is either partially or totally replaced with donor cadevar corneal tissue. The surgery is typically performed on an outpatient basis under local injectable anesthesia. Although there is some risk of rejection, the corneal is essentially avascular, which reduces such risk. Candidates for keratoplasty or penetrating keratoplasty include patient with keratoconus (inherited corneal thinning) with visual distortion, with Fuch's dystrophy (corneal clouding, either heriditary or acquired) with vision loss, and/or corneal scarring (e.g., due to injury). Full visual recovery takes up to a year post-surgery.
Corneal sensation is reduced in each of the described procedures; corneal sensation is reduced to a greater extent in procedures that involve cutting compared to procedures that do not involve cutting.
Upon completion of any of these procedure, the duration typically being less than one minute, medicaments, typically anti-inflammatory agents and/or antibiotics, are topically applied to the corneal surface. In procedures that involve cutting, a contact lens is then placed on the eye.
An example of a medication administration protocol used in the inventive method follows. At the completion of the LASIK procedure, one drop of cyclosporine (0.05% w/v) composition, such as a liquid solution or emulsion, was administered onto the corneal surface(s) of the eye(s). After at least ten minutes one drop of an anti-inflammatory agent (e.g., prednisolone) and one drop of an antibacterial agent (e.g., moxifloxacin) were administered to the eye. This administration protocol was followed hourly on the day of surgery while the patient was awake. An ocular lubricant was also administered to the corneal surface hourly after surgery and then as needed to alleviate dry eye sensation. Other suitable anti-inflammatories, antiirritants, antibiotics, and ocular lubricants may be used, as known to one skilled in the art.
On the day after surgery one drop of cyclosporin (0.05% w/v) composition was administered to the eye twice a day at intervals of about 12 hours. Then, for up to about six months or more depending upon the progress of recovery, a drop of the antiinflammatory/antiirritant and/or antibiotic was administered to the eye four times daily for one week and then twice a day for two days, and then once a day for two days. The medications were administered to the eye before a lens was placed in the eye. The eye lubricant was uses on an as needed basis. The protocol was varied according the progress of recovery, the sensitivity of the patient to the medications, and other factors as known to one skilled in the art.
One drop of drop of cyclosporine (0.05% w/v) composition (liquid solution or emulsion) has a volume of about 0.065 ml (65 μl), which would contain about 32.5 μg cyclosporine. Solutions of cyclosporine may have concentrations ranging from about 0.01% w/v to about 1.0% w/v cyclosporine, thus the amount of cyclosporine that can be administered in a drop of solution to enhance ocular neuroprotection ranges from about 6.5 μg to about 650 μg. In other embodiments the concentration of cyclosporine ranged from about 0.05% to about 0.5% and 0.05% to about 0.1%, resulting in amounts of cyclosporine administered from about 6.5 μg to about 325 μg and from about 6.5 μg to about 65 μg. One skilled in the art will know the amount of cyclosporine delivered will vary with the volume (size) of the drop of solution which is determined by physiochemical factors such as the number of components in solution or emulsion (i.e., excipients, surfactants, chelators, etc.), surface tension, aperture size of delivery apparatus, etc. Solutions of cyclosporine are commercially available (e.g., RESTASIS® (0.05% cyclosporine ophthalmic emulsion, Allergen).
Patients were typically evaluated at post-surgical intervals of one day, three days, one week, one month, two to three months, and four to six months. The bandage contact lens was typically removed on the second or third post-operative day when the epithelium had healed. Most patients required eye drops to control healing only during the first six to twelve weeks, but other patients, usually those with greater refractive error, required topically applied medicaments for up to six months or more following surgery.
After any of these corneal surgeries, patients may experience problems relating to the loss of ocular sensitivity or sensation. For example, decreased ocular nerve function makes the cornea prone to trauma, which in turn can lead to infection. It reduces the usual blink mechanism that is required to keep the corneal surface moist, leading to drying and sloughing of the corneal epithelium. This, in turn, causes cloudiness of the flap, prones the flap to infection by enteral pathogens because of loss of barrier, and reduces vision.
One embodiment of the invention is a method for ameliorating or restoring loss of corneal sensation, enhancing ocular nerve regeneration, and/or restoring nerve function (re-enervation) in the eye of a patient by providing locally to the eye a composition comprising at least one neuroprotective and/or neurostimulatory factor. Another embodiment is a method for prophylactically administering, to an eye of a patient at risk for an ocular neurologic and/or neurosensory pathology, a neuroprotective and/or neurostimulatory factor to ameliorate or restore loss of corneal sensation, enhance ocular nerve regeneration, and/or provide reeneravation.
One embodiment of the invention locally administers one or more agents that enhance corneal sensation, possibly by nerve regeneration and/or enervation. In one embodiment, one or a combination of macrolides, including macrolide analogues, is administered, the macrolide and/or analogue having neuro-stimulatory activity. In another embodiment, one or a combination of macrolides is administered with one or more agent(s) that enhance corneal nerve stimulation. Such neurostimulatory agents may increase nerve cell quantity, functional quality, or combinations of these. One skilled in the art will appreciate that enhancement refers to any qualitative and/or quantitative improvement in corneal sensation and/or ocular neurological function following surgery regardless of degree.
One embodiment of the invention is a composition containing a neurostimulatory or neuroprotective macrolide, macrolide analog, neurotrophin, and/or neuropoietic factor that may be administered prophylactically to patients having or at risk for developing glaucoma, retinitis pigmentosa, or other neurosensory or neurodegenerative disease, or may be administered to patients with glaucoma or retinitis pigmentosa, either alone or in conjunction with other therapy.
Glaucoma is a general term for several types of a painless ocular condition that, left untreated, can result in partial or complete vision loss. It is characterized by elevated intraocular pressure, considered by one skilled in the art as a pressure greater than about 21.5 mm Hg. The higher the intraocular pressure, the greater the likelihood of optic nerve damage and visual field loss. In glaucoma monitoring or therapy, the neurodegenerative component should be considered in addition to therapy for increased intraocular pressure, such as protection of retinal ganglion cells (RGC).
Known risk factors for glaucoma include age (elevated risk for individuals over age 60), race (elevated risk for African Americans over age 40), a family history of glaucoma, individuals with diabetes, severe nearsightedness, long-term corticosteroid use, previous eye injury, and/or increased intraocular pressure. One risk factor may suffice for prophylactic administration of a neuroprotective and/or neurostimulatory agent as described herein, and risk factors may alter over time, as known to one skilled in the art.
In monitoring, diagnosing, and/or treating patients with glaucoma, attainment of decreased intraocular pressure is a necessary but insufficient goal. This is because a component of glaucoma is neurologic damage to the optic nerve and ganglion cell death, so that its neurodegenerative aspects must be considered. Even patients with normal intraocular pressure may develop glaucoma-like changes. Further, retinal ganglion cells may be more sensitive to increased intraocular pressure, whereas other ocular cells may be better able to withstand increased intraocular pressure.
Retinitis pigmentosa is a general term that encompasses a disparate group of disorders of rods and cones. Because retinitis pigmentosa affects these retinal sensory structures, prophylactic or therapeutic administration of neuroprotective or neurostimulatory agents may reduce decreased visual field and other adverse effects.
In one embodiment, a patient is prophylactically or therapeutically administered a neurostimulatory and/or neuroprotective macrolide, macrolide analog, neurotrophin, and/or neuropoietic agent. The inventive method may prevent or delay an increase in intraocular pressure, may reduce associated nerve loss, may confer protection on retinal sensory cells, etc. Administration may be by any ocular route. One example is topical application, with the neurostimulatory and/or neuroprotective agent(s) administered in a formulation of eye drops, cream, ointment, gel, salve, etc. Another example is intraocular injection with the neurostimulatory and/or neuroprotective agent administered subconjunctivally, intravitreally, retrobulbarly, within the crystalline lens via piercing the lens capsule as described in co-pending U.S. patent application Ser. No. 11/103,283 which is expressly incorporated by reference herein, etc. Another example provides the neurostimulatory and/or neuroprotective agent to the eye on or in a formulation such as a liposome, microsphere, microcapsule, biocompatible matrix, gel, polymer, nanoparticle, nanocapsule, etc. Another example provides the neurostimulatory and/or neuroprotective agent on or in a device such as a device for transscleral delivery as described in co-pending U.S. patent application Ser. No. 11/105,756, or another intraocular device using, for example, iontophoresis or another type of release mechanism (controlled or not controlled), as known by one skilled in the art. Another example provides the neurostimulatory and/or neuroprotective agent in conjunction with gene therapy, as known by one skilled in the art.
A topical formulation may be administered by any ophthalmogical vehicle, as know to one skilled in the art. Examples include, but are not limited to, eye droppers, satchels, applicators, etc. The amount and concentration of the formulation may depend upon the diluent, delivery system or device selected, clinical condition of the patient, side effects expected, stability of the compounds of the composition, presence and severity of other pathology, dosing frequency, active agent, etc.
For topical administration, examples of concentrations that may be used include but are not limited to, less than 1 μg/mL, 1 μg/mL to 5 μg/mL, 5 μg/mL to 10 μg/mL, 10 μg/mL to 50 μg/mL, 50 μg/mL to 100 μg/mL, 100 μg/mL to 0.5 mg/mL, 0.5 mg/mL to 2.5 mg/mL, 1 mg/mL to 5 mg/mL, 5 mg/mL to 10 mg/mL, 10 mg/mL to 15 mg/mL, 15 mg/mL to 30 mg/mL, and greater than 30 mg/mL. For topical administration, examples of dosing regimens that may be used include but are not limited to, hourly, half-daily, daily, weekly, biweekly, monthly, quarterly, three times a year, twice a year, yearly, every two years, every three years, etc. Intervals between doses may be regular or varied. As one example, doses may be administered hourly or daily pre- and post-surgery for one week, for several weeks, or for several months, then may be administered twice a year or once a year until the desired neurostimulation and/or neuroprotection is achieved. As another example, doses may be administered daily or weekly pre- and/or post-surgery for one week, for several weeks, for several months, or for several years until the desired neurostimulation and/or neuroprotection is achieved.
Ganglion cells in the retina (RGC) that have been damaged (e.g., by elevated intraocular pressure) undergo apoptosis, also referred to as programmed cell death. The macrolide tacrolimus, systemically administered, conferred neuroprotection on RGC by interfering with apoptotic mechanisms, as disclosed in Freeman and Grosskreutz Investigative Ophthalmology & Visual Science:41, 1111 (2000), which is expressly incorporated by reference herein in its entirety. As a result of programmed cell death, RGC release compounds whose presence and/or concentration may result in toxicity, remove desirable agents and/or alter cell signaling; these compounds include cytokines, the excitatory neurotransmitter glutamate, Ca2+ binding proteins, FK 506 (tacrolimus) binding proteins, and others. Thus, ocular administration of a neurostimulatory and/or neuroprotective macrolide, macrolide analog, neurotrophin, and/or neuropoietic factor may reduce or inhibit subsequent effects of the released cytokines, glutamate, etc. that are part of the neurodegenerative processes associated with glaucoma and/or retinitis pigmentosa. For example, macrolides tacrolimus and cyclosporine are potent immunosuppresants that inhibit T-cell activation by interfering with signal transduction. In vitro, tacrolimus binds to and inhibits the activity of the immunophilin FK 506-binding protein (FKBP), an isomerase that functions in signal transduction and cell communication. Reducing apoptotic mechanisms would reduce such processes, and thus protect or delay neurosensory impairment or neurodegenerative damage.
Such administration of macrolides may be alone or may be in conjunction with other agents used to reduce intraocular pressure in patients with elevated intraocular pressure due to ocular hypertension or open-angle glaucoma. For example, administration may be included with a current drug regimen, or at different intervals than a current regimen, or for a set duration, etc; all these are examples of administration in conjunction with other agent. Examples of known drugs include, but are not limited to, Diamox® (acetazolamide (N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide), an inhibitor of carbonic anhydrase, Wyeth, Madison N.J.); Timoptic® (timolol maleate ophthalmic solution, Merck, Whitehouse Station N.J.), Xalatan® (latanaprost ophthalmic solution, Pfizer, Groton Conn.); Copaxone® (Teva Pharmaceuticals, Petah Tiqva, Israel); Memantine (Allergan, Irvine Calif.); Alphagan® P (brimonidine tartrate ophthalmic solution; Allergan); and others known to one skilled in the art. In this embodiment, the inventive method may use macrolides to potentiate the action of current treatments. For example, acetazolamide may assist in normal polarization of cell membranes, so the effect of local ocular treament with acetazolamide and a neuroprotective macrolide or agent, is reduced apoptic effects and normalized polarization of sensory retinal ganglion cells. The dual action may be additive or synergistic.
Macrolides encompassed by the invention are those known by one skilled in the art, as well as analogs and derivatives. These are disclosed in, for example, co-pending U.S. patent application Ser. Nos. 10/667,161 and 10/752,124. Macrolides and their analogues that may be administered include the following.
Cyclosporine (Cyclosporin A, topical formulation Arrestase®, Allergan Inc.) is a cyclic peptide produced by Trichoderma polysporum. It is available commercially, for example, from Sigma-Aldrich (St. Louis Mo.). It is an immunosuppressant and acts in a particular subset of T lymphocytes, the helper T cells. Cyclosporine exerts an immunosuppressant effect by inhibiting production of the cytokine interleukin 2. Each of cyclosporine and tacrolimus, another immunosuppressant, produces significant renal and hepatic toxicity when each is administered systemically; because of this toxicity, they are not administered together. The use of cyclosporine as a specific medicament for treatment of ocular disease with reduced toxicity is described in co-pending U.S. patent application Ser. No. 10/289,772. Cyclosporine is commercially available as a 0.05% emulsion for ophthalmic application under the trademark RESTASIS® (Allergan). Its indication is increased tear production in patients with ocular inflammation associated with keratoconjunctivitis sicca (chronic dry eye).
Tacrolimus (Prograf®, previously known as FK506), a macrolide immunosuppressant produced by Streptomyces tsukubaensis, is a tricyclo hydrophobic compound that is practically insoluble in water, but is freely soluble in ethanol and is very soluble in methanol and chloroform. It is available under prescription as either capsules for oral administration or as a sterile solution for intravenous administration. The solution contains the equivalent of 5 mg anhydrous tacrolimus in 1 ml polyoxyl 60 hydrogenated castor oil (HCO-60), 200 mg, and dehydrated alcohol (USP, 80.0% v/v), and must be diluted with a solution of 0.9% NaCl or 5% dextrose before use.
Sirolimus, also known as rapamycin, RAPA, and Rapamune®, is a triene macrolide antibiotic derived from Streptomyces hydroscopicus and originally developed as an antifungal agent. Subsequently, it has shown anti-inflammatory, anti-tumor, and immunosuppressive properties. Pimecrolimus, also known as ascomycin, Immunomycin, and FR-900520, is an ethyl analog of tacrolimus and has strong immunosuppressant properties. It inhibits Th1 and Th2 cytokines, and preferentially inhibits activation of mast cells, and is used to treat contact dermatitis and other dermatological conditions. Sirolimus and pimecrolimus are commercially available, e.g., A.G. Scientific, Inc. (San Diego Calif.).
Regarding its immunosuppressive potential, sirolimus has some synergetic effect with cyclosporine. It has been reported that sirolimus has a different mode of action compared to cyclosporine and tacrolimus. All three agents are immunosuppressants which affect the action of immune cell modulators (cytokines), but do not affect the immune cells themselves. However, while all three agents affect immune cell modulators, they do so differently: cyclosporine and tacrolimus prevent synthesis of cytokine messengers, specifically interleukin-2, while sirolimus acts on cytokine that has already been synthesized, preventing it from reaching immune cells.
Sirolimus inhibits inflammation by acting on both T-lymphocytes and dendritic cells. The latter are the first cells to recognize antigens. Sirolimus blocks the growth of dendritic cells and a number of other cells, such as tumors and endothelial cells, which are activated by the tumor cell releasing vascular endothelial growth factor (VEGF). VEGF is a central regulator of angiogenesis (formation of new blood vessels from pre-existing vessels) and vasculogenesis (development of embryonic vasculature through an influence on endothelial cell differentiation and organization). Diseases that are characterized by abnormal angiogenesis and vasculogenesis, such as some cancers and some ocular diseases, may show abnormal production of VEGF. Thus, control of VEGF function may be one means to control or treat these diseases. Sirolimus has also been used in the prevention of smooth muscle hyperplasia after coronary stent surgery. The use of sirolimus and ascomycin as specific medicaments for treatment of ocular disease has been disclosed in co-pending U.S. patent application Ser. No. 10/631,143.
Everolimus, also known as RAD-001, SCZ RAD, Certican® (Novartis, Basel Switzerland), is an analog of sirolimus but is a new and distinct chemical entity. It is an oral immunosuppressant that inhibits growth factor-induced cell proliferation and thus reduces acute organ rejection and vasculopathy, the proliferation of smooth muscle cells in the innermost wall of grafts that restricts blood supply.
It will be appreciated that the invention encompasses the use of macrolides in addition to those previously described. These include, for example, clindamycin, leucomycins, zoatrolimus, the known antibiotics erythromycin and its derivatives such as azithromycin and clarithromycin, lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin, troleandomycin, tylosin, and roxithromycin, and other macrolides such as biolimus, ABT-578 (methylrapamycin); macrolide derivatives such as temsirolimus (CCI-779, Wyeth) and AP23573 (Ariad) (both rapamycin derivatives). The invention also includes new macrolide antibiotic scaffolds and derivatives in development, including but not limited to the ketolides ABT-773 and telithromycin as described by Schonfeld and Kirst (Eds.) in Macrolide Antibiotics, Birkhauser, Basel Switzerland (2002); macrolides derived from leucomycins, as described in U.S. Pat. Nos. 6,436,906; 6,440,942; and 6,462,026 assigned to Enanta Pharmaceuticals (Watertown Mass.); and lincosamides, each of which is incorporated by reference herein.
Any of the above-described macrolides may be used in the invention. In one embodiment, the total macrolide concentration ranges from less than 1 ng/ml to about 10 mg/ml. In another embodiment, the total macrolide concentration ranges from about 1 ng/ml to about 1 mg/ml. In another embodiment, the total macrolide concentration ranges from about 20 μg/ml to about 200 μg/m. In another embodiment, the total macrolide concentration is below 5 mg/ml. Formulations and doses of macrolides are described in U.S. patent application Ser. Nos. 10/667,161 and 10,/752/124, each of which is expressly incorporated by reference herein in its entirety.
Specific macrolide analogues accelerate nerve regeneration and functional recovery, as disclosed in Revill et al., J. Pharmacol. Exp. Therap. (2002) 302; 1278, which is expressly incorporated by reference herein in its entirety. For example, genetically engineered 13- and 15-desmethoxy analogs of ascomycin, examples of macrolide analogs, that contain hydrogen, methyl, or ethyl instead of methoxy at either the 13-, the 15-, or both the 13- and 15-positions, enhanced neurite outgrowth in cultured SH-SY5Y neuroblastoma cells at concentrations of 1 mg/kg/day and 5 mg/kg/day, with nerve growth factor (NGF) at a concentration of 10 ng/ml. The ascomycin analog 13-desmethoxy-13-methyl-18 hydroxy (13-Me-18-OH), at concentrations of 1 mg/kg/day and 5 mg/kg/day, was demonstrated to accelerate nerve regeneration and lead to full functional recovery (walking) in a rat sciatic nerve crush model.
The combination of a macrolide and another neurostimulatory or neuroprotective factor(s) such as neurotrophins or neuropoietins is used in one embodiment.
Neurotrophins are a family of polypeptides that enhance survival of nervous tissue by maintenance, growth, differentiation, etc. They stimulate the growth of sympathetic and sensory nerve cells in both the central and peripheral nervous system. All neurotrophins have six conserved cysteine residues and share a 55% amino acid sequence identity. Some are in a pro-neurotrophin form and are cleaved to produce a mature form. Examples of neurotrophins include nerve growth factor-β (NGFβ), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotrophin 6 (NT-6). These are available commercially, for example, from Sigma-Aldrich (St. Louis Mo.); Axxora (San Diego Calif.) mouse 2.5S and 7S components NGFβ, human recombinant β-NGF and pro-β-NGF. Further examples of neurotrophins are products of the neural regeneration protein (NRP) gene.
Different neuron types require different neurotrophins, depending upon their receptor expression. All neurotrophins are capable of binding to p75 neurotrophin growth factor receptors, which are low affinity receptors. Specific neurotrophins and mature neurotrophins bind to different tyrosine kinase (trk) receptors, which are higher affinity receptors than p75 receptors. Tyrosine kinase receptors include types A (trkA), B (trkB), and C (trkC).
NGFβ is a specific ligand for the trkA receptor and signals through trkA. It also signals through the low affinity p75 receptors. NGFβ is a secreted protein that helps to develop and maintain the sympathetic nervous system, affecting sensory, pain, and sympathetic targets. It is required for survival of small, peptide-expressing neurons that express the trkA receptor and that project into the superficial laminae of the dorsal horn (i.e., putative nociceptive neurons).
BDNF signals through trkB, in addition to the low affinity p75 receptors. It is Ca2+ dependent and may control synaptic transmission and long term synaptic plasticity, affecting sensory and motor targets. It enhances survival and differentiation of several classes of neurons in vitro, including neural crest and placode-derived sensory neurons, dopaminergic neurons in the substantia nigra, basal forebrain cholinergic neurons, hippocampal neurons, and retinal ganglial cells. BDNF is expressed within peripheral ganglia and is not restricted to neuronal target fields, so that it may have paracrine or autocrine actions on neurons as well as non-neuronal cells.
Neurotrophin-3 (NT-3) is part of the family of neurotrophic factors that control survival and differentiation of mammalian neurons. NT-3 is closely related to NGFβ and BDNF. The mature NT-3 peptide is identical in all mammals examined including human, pig, rat and mouse. NT-3 preferentially signals through trkC, over trkA and trkB receptors, and also utilizes the low affinity p75 receptors. It functions at the neuromuscular junction, affecting large sensory and motor targets and regulating neurotransmifter release at neuromuscular synapses. It may be involved in maintenance of the adult nervous system, and affect development of neurons in the embryo when it is expressed in human placenta.
Neurotrophin 4 (NT-4, synonymous with NT-5) belongs to the NGF-β family and is a survival factor for peripheral sensory sympathetic neurons. NT-4 levels are highest in the prostate, with lower levels in thymus, placenta, and skeletal muscle. NT-4 is also expressed in embryonic and adult tissues. It signals through trkB in addition to low affinity p75 receptors, affecting sympathetic, sensory, and motor targets. Neurotrophin-6 has also been reported.
Ciliary neurotrophic factor (CNTF) is expressed in glial cells within the central and peripheral nervous systems. It stimulates gene expression, cell survival, or differentiation in a variety of neuronal cell types such as sensory, sympathetic, ciliary, and motor neurons. CNTF itself lacks a classical signal peptide sequence of a secreted protein, but is thought to convey its cytoprotective effects after release from adult glial cells by some mechanism induced by injury. In addition to its neuronal actions, CNTF also acts on non-neuronal cells such as glia, hepatocytes, skeletal muscle, embryonic stem cells, and bone marrow stromal cells.
Glial cell derived neurotrophic factor (GDNF) is a 20 kD glycosylated polypeptide that exists as a homodimer. It stimulates the growth of dopaminergic neurons and autonomic motor neurons.
Neuropoietic factors may be used in addition to, or in place of, neurotrophic factors. Neuropoietic factors regulate the properties of cells both in the peripheral and central nervous systems, and both during development and in the mature nervous system. They regulate neuronal phenotype (neurotransmitter) and differentiation of neuronal precursor cells in peripheral and spinal cord neurons. They also regulate cell survival, and development of astrocytes and oligodendrocytes. Neuropoietic factors are also trauma factors in rescuing sensory and motor neurons from axotomy-induced cell death. They show temporal and spatial specific expression patterns, and have specific roles in neural development and repair.
Neuropoietic factors include some cytokines, different from cytokines associated with apoptosis-induced neurodegenerative processes, and hematopoietic factors that fulfill criteria for demonstrating a role in neuronal differentiation and survival. They include leukemia inhibitory factor (LIF), oncostatin M, growth-promoting activity, and cardiotrophin 1. All of these factors activate a subfamily of class I cytokine receptors, the interleukin-6 receptor family.
Any of the above-described neurotrophins and/or neuropoietic factors may be used in the invention. In one embodiment, the total concentration of neurotrophins and/or neuropoietic factors ranges from about 1 pM to about 100 pM. In another embodiment, the total concentration of neurotrophins and/or neuropoietic factors ranges from about 0.01 nM to about 1 M. In another embodiment, the total concentration of neurotrophins and/or neuropoietic factors is below 1 nM. The neurotrophin(s) and/or neuropoietic factor(s) may be used singly or in combination.
The addition of a macrolide, macrolide analog, neurotrophin and/or a neuropoietic factor, alone or in combination, in an ocular formulation, provides beneficial results in enhancing corneal sensation, nerve regeneration, protection, and/or re-enervation. In embodiments where a macrolide is present, the composition also reduces post ocular surgical scarring, provides anti-inflammatory effects, and may provide anti-infective properties. It will be appreciated that various embodiments are contemplated. As one example, a macrolide or macrolide analog, with or without neurostimulatory activity, may be used without a neurotrophin or neuropoietic factor. As another example, a neurotrophin or neuropoietic factor or any other neuro-stimulatory factor or factors may be used alone. As another example, other agents may be included in the composition. Examples of these agents include, but are not limited to, steroids, non-steroidal anti-inflammatory agents (NSAIDS), antibiotics, antioxidants, anti-proliferative, anti-cell migration, and/or anti-angiogenic agents.
Steroids for ocular administration include, but are not limited to, triamcinolone (Aristocort®; Kenalog®), betamethasone (Celestone®), budesonide, cortisone, dexamethasone (Decadron-LA®; Decadron® phosphate; Maxidex® and Tobradex® (Alcon)), hydrocortisone, methylprednisolone (Depo-Medrol®, Solu-Medrol®), prednisolone (prednisolone acetate, e.g., Pred Forte® (Allergan); Econopred and Econopred Plus® (Alcon); AK-Tate® (Akorn); Pred Mild® (Allergan); prednisone sodium phosphate (Inflamase Mild and Inflamase Forte® (Ciba); Metreton® (Schering); AK-Pred® (Akorn)), fluorometholone (fluorometholone acetate (Flarex® (Alcon); Eflone®), fluorometholone alcohol (FML® and FML-Mild®, (Allergan); FluorOP®)), rimexolone (Vexol® (Alcon)), medrysone alcohol (HMS® (Allergan)); lotoprednol etabonate (Lotemax® and Alrex® (Bausch & Lomb), 11-desoxcortisol, and anacortave acetate (Alcon)).
Antibiotics include, but are not limited to, doxycycline (4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1, 11-dioxo-2-naphthacenecarboxamide monohydrate, C22H24N2O8.H2O), aminoglycosides (e.g., streptomycin, amikacin, gentamicin, tobramycin), cephalosporins (e.g., beta lactams including penicillin), tetracyclines, acyclorvir, amantadine, polymyxin B, amphtotericin B, amoxicillin, ampicillin, atovaquone, azithromycin, azithromycin, bacitracin, cefazolin, cefepime, cefotaxime, cefotetan, cefpodoxime, ceftazidime, ceftizoxime, ceftriaxone, cefuroxime, cephalexin, chloramphenicol, clotimazole, ciprofloxacin, clarithromycin, clindamycin, dapsone, dicloxacillin, erythromycin, fluconazole, foscarnet, ganciclovir, gatifloxacin, griseofulvin, isoniazid, itraconazole, ketoconazole, metronidazole, nafcillin, neomycin, nitrofurantoin, nystatin, pentamidine, rifampin, rifamycin, valacyclovir, vancomycin, etc.
Anti-proliferative agents include, but are not limited to, carboplatin, 5-fluorouracil (5-FU), thiotepa, etoposide (VP-16), doxorubicin, ifosphophamide, cyclophosphamide, etc.
Anti-prostaglandins include, but are not limited to, indomethacin, ketorolac tromethamine 0.5% ((±)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid, compound with 2-amino-2-(hydroxymethyl)-1,3-propanediol (1:1) (ACULAR® Allegan, Irvine Calif.), OCUFEN® (flurbiprofen sodium 0.03%), meclofenamate, fluorbiprofen, and compounds in the pyrrolo-pyrrole group of non-steroidal anti-inflammatory drugs.
A matrix metalloproteinase inhibitor may be added. These include, but are not limited to, doxycycline, TIMP-1, TIMP-2, TIMP-3, TIMP-4, MMP1, MMP2, MMP3, Batimastat, or marimastat. Eight matrix metalloproteinase inhibitors have been identified in the cornea, namely, collagenases I and III (MMP-1 and MMP-13), gelatinases A and B (MMP-2 and MMP-9), stromelysin (MMP-3), matrilysin (MMP-7), and membrane type MMP (MMP-14).
Anti-angiogenesis agents include, but are not limited to, antibodies to vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab) (Genentech), and other anti-VEGF compounds; MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals), pigment epithelium derived factor(s) (PEDF); CELEBREX®; VIOXX®; interferon alpha; interleukin-12 (IL-12); thalidomide and derivatives such as REVIMID™ (CC-5013) (Celgene Corporation); squalamine; endostatin; angiostatin; the ribozyme inhibitor ANGIOZYME® (Sirna Therapeutics); multifunctional antiangiogenic agents such as NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada); etc., as known to one skilled in the art.
Other agents may also be added, such as NSAIDS, vitamins, minerals, cytokines, growth factors, etc. Examples of the above include, but are not limited to, colchicine, naproxen sodium (ANAPROX® and ANAPROX DS®, (Roche); flurbiprofen (ANSAID®, Pharmacia Pfizer); diclofenac sodium and misoprostil (ARTHROTEC®, Searle Monsanto); valdecoxib (BEXTRA®, Pfizer); diclofenac potassium (CATAFLAM®, Novartis); celecoxib (CELEBREX®, Searle Monsanto); sulindac (CLINORIL®, Merck); oxaprozin (DAYPRO®, Pharmacia Pfizer); salsalate (DISALCID®, 3M); salicylate (DOLOBID®, Merck); naproxen sodium (EC NAPROSYN®, Roche); piroxicam (FELDENE®, Pfizer); indomethacin (INDOCIN®, Merck); etodolac (LODINE®, Wyeth); meloxicam (MOBIC®, Boehringer Ingelheim); ibuprofen (MOTRIN®, Pharmacia Pfizer); naproxen (NAPRELAN®, Elan); naproxen (NAPROSYN®, Roche); ketoprofen (ORUDIS®, ORUVAIL®, Wyeth); nabumetone (RELAFEN®, SmithKline); tolmetin sodium (TOLECTIN®, McNeil); choline magnesium trisalicylate (TRILISATE®, Purdue Fredrick); rofecoxib (VIOXX®, Merck), vitamins A, B (thiamine), B6 (pyridoxine), B12 (cobalamine), C (ascorbic acid), D1, D2 (ergocalciferol), D3 (cholcalciferol), E, K (phytonadione), K1 (phytylmenaquinone), K2 (multiprenylmenaquinone); carotenoids such as lutein and zeaxanthin; macrominerals and trace minerals including, but not limited to, calcium, magnesium, iron, iodine, zinc, copper, chromium, selenium, manganese, molybdenum, fluoride, boron, etc. Commercially available supplements are also included such as high potency zinc (commercially available as OCUVITE® PRESERVISION®, Bausch & Lomb, Rochester N.Y.), or high potency antioxidants (zinc, lutein, zeaxanthin) (commercially available as ICAPS® Dietary Supplement, Alcon, Fort Worth Tex.).
It will be appreciated that the above agents include pharmaceutically acceptable salts and derivatives (e.g., sodium, potassium bicarbonate, sulfate, etc.), and are representative but not exclusive. The duration over which any of the formulation will dwell in the ocular environment will depend on factors that include, but are not limited to, compound(s) pharmacological properties, concentrations, and/or bioavailability, disease, mode of administration, desired longevity of therapy, etc. Formulations of embodiment may have dwell times of hours, days, weeks, months, or years.
In one example, rabbits are administered neurostimulatory or neuroprotective macrolides, macrolide analogs, neurotrophins, and/or neuropoietic factors (treated) or vehicle alone (control). To generate damage to retinal sensory or ganglion cells, anesthetized rabbits may be subjected to a sever crush injury of the optic nerve, or may be treated to induce increased intraocular pressure, or may be treated to induce retinal ischemic/reperfusion injury, or may be subject to other methods known to one skilled in the art.
As an example of one embodiment, after excising the conjunctiva and exposing the optic nerve with the aid of a binocular operating microscope with care not to interfere with the blood supply, the nerve can be mechanically crushed for a defined period using forceps or other instruments, as described in Schori et al., PNAS 98:3398(2001), which is expressly incorporated by reference herein in its entirety.
As an example of another embodiment, rabbits may be treated to result in intraocular pressure greater than 17 mm Hg. This may be done by negative pressure applied to a corneoscleral ring fixed to the sclera and connected to a vacuum source, as known to one skilled in the art. This may also be done by positive pressure applied through a cannula connected to the interior chamber. This may also be done by blocking aqueous outflow using 80-120 applications of blue-green argon laser as described in Bakalash et al., Investigative Ophthalmology & Visual Science 44: 3374 (2003), which is expressly incorporated by reference herein.
As an example of another embodiment, the central and choroidal arteries may be surgically closed. Deprivation of blood flow to the retina would result in ischemia due to lack of oxygen and nutrients, while reperfusion would result in free radical injury; this type of ischemia/reperfusion injury is known to one skilled in the art.
One or a combination of the macrolides, macrolide analogues, neurotrophins, and neuropoietic factors in different combinations of agent, dose, route of administration, intervals, etc. as described herein may be used and administered as previously described.
Assessment of retinal damage in control and treated animals may be by applying dextran tetramethylrhodamine, a hydrophilic neurotracer (Molecular Probes, Eugene Oreg.) into the intraorbital portion of the optic nerve, with only functional axons capable of dye uptake. Rabbits are sacrificed twenty-four hours after dye administration, retinas excised, wholemounted, and preserved in 4% paraformaldehyde. Retinal ganglial cells are counted under 800× magnification using a fluorescence microscope. Four fields from each retina are counted with the same diameter and located the same distance from the optic disc. Eyes from untreated rabbits are used as controls.
Other dyes or markers for viable ganglion cells can be introduced and the number of cells can be counted in treated groups versus control groups. In addition, factors other than dye uptake can be used as an indicator of neuroprotection and/or neurostimulation. These factors include retinal ganglial and/or sensory cell morphology from treated versus control groups, assays of cellular function, conductivity, etc. Conversely, apoptosis may be assayed in retinal ganglial and/or sensory cells from treated versus control groups. For example, Annexin V binding is known by one skilled in the art as an indirect indicator of apoptosis, and binding can be assayed in treated versus control cells. A clonagenic assay is known by one skilled in the art as a direct indicator of apoptosis, and can be performed with the results compared from both treated and control cells.
It will be appreciated that the agents include pharmaceutically acceptable salts and derivatives thereof (e.g., sodium, potassium, bicarbonate, sulfate, etc). It will also be appreciated that the above lists are representative only and are not exclusive. The indications, effective doses, formulations (including buffers, salts, and other excipients), contraindications, vendors, etc. of each of the above are known to one skilled in the art.
In one embodiment, the composition is formulated for topical application. In another embodiment, the composition is formulated for intraocular application. In another embodiment, the composition is formulated for subconjunctival or intravitreal application. In another embodiment, the composition is in a delayed- or extend-release formulation. In another embodiment, the composition is formulated on or in an intraocular lens (e.g., implanted lens, contact lens). In another embodiment, the composition is formulated on or in an implanted ocular device. None of these formulations result in significant systemic absorption, so that there are no detrimental effects that may result with systemically administered macrolides and/or neurostimulatory factor(s).
The formulation may be a slow, extended, or time release formulation, a carrier formulation such as microspheres, microcapsules, liposomes, etc., as known to one skilled in the art. Any of the above-mentioned delayed release delivery systems may be administered topically, intraocularly, subconjunctivally, or by implant to result in sustained release of the agent over a period of time. The formulation may be in the form of a vehicle, such as a micro- or macro-capsule or matrix of biocompatible polymers such as polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and other polymers such as those disclosed in U.S. Pat. Nos. 6,667,371; 6,613,355; 6,596,296; 6,413,536; 5,968,543; 4,079,038; 4,093,709; 4,131,648; 4,138,344; 4,180,646; 4,304,767; 4,946,931, each of which is expressly incorporated by reference herein in its entirety, or lipids that may be formulated as microspheres or liposomes. A microscopic or macroscopic formulation may be administered topically or through a needle, or may be implanted. Delayed or extended release properties may be provided through various formulations of the vehicle (coated or uncoated microsphere, coated or uncoated capsule, lipid or polymer components, unilamellar or multilamellar structure, and combinations of the above, etc.). The formulation and loading of microspheres, microcapsules, liposomes, etc. and their ocular implantation are standard techniques known by one skilled in the art, for example, the use a ganciclovir sustained-release implant to treat cytomegalovirus retinitis, disclosed in Vitreoretinal Surgical Techniques, Peyman et al., Eds. (Martin Dunitz, London 2001, chapter 45); Handbook of Pharmaceutical Controlled Release Technology, Wise, Ed. (Marcel Dekker, New York 2000), the relevant sections of which are incorporated by reference herein in their entirety. For example, a sustained release intraocular implant may be inserted through the pars plana for implantation in the vitreous cavity. An intraocular injection may be into the vitreous (intravitreal), or under the conjunctiva (subconjunctival), or behind the eye (retrobulbar), or under the Capsule of Tenon (sub-Tenon), and may be in a depot form. The composition may be administered via a contact lens applied to the exterior surface of an eye, with the composition incorporated into the lens material (e.g., at manufacture, or contained in a lens solution). The composition may be administered via an intraocular lens (IOL) that is implanted in the eye. Implantable lenses include any IOL used to replace a patient's diseased lens following cataract surgery, including but not limited to those manufactured by Bausch and Lomb (Rochester N.Y.), Alcon (Fort Worth Tex.), Allergan (Irvine Calif.), and Advanced Medical Optics (Santa Ana Calif.). When the lens is implanted within the lens capsule, the composition provides the desired effect to the eye. Concentrations suitable for implants (lenses and other types) and by contact lens administration may vary, as will be appreciated by one skilled in the art. For example, an implant may be loaded with a high amount of agent, but formulated or regulated so that a required concentration within the above-described ranges is sustainedly released (e.g., slow release formulation).
In various embodiments, the composition is administered up to four times a day. In embodiments where the composition is administered after surgery, administration may commence following surgery on the same day, or the day after surgery, or a few days after surgery, or any time after surgery. The composition may be self-administered or administered by another, for example, if visual acuity is poor, or if the patient is uncomfortable with self-administration. The patient is periodically evaluated (e.g., daily, every other day, etc.) using assessment methods known to one skilled in the art. In embodiments where the composition is used to assess corneal sensation, these include assessment of corneal clarity, corneal sensation (e.g., using a Cochet-Bonnet filament-type aesthesiometer), corneal enervation, etc. In embodiments where the composition is used to enhance ocular neuroprotection and/or neurostimulation, these may include one or more of the following assessments: retinal ganglial cell viability, quantitation of ocular glutamate levels, visual field and visual acuity determinations, assessment of visual evoked potential (VEP) to evaluate visual neural pathways via electrode measurement of brain electrical activity while watching a moving pattern on a video monitor, electroretinogram (ERG) to evaluate the ocular electrical responses to a flash of light using an electrode placed on the surface of the eye (e.g., cornea), electrooculargram (EOG), critical flicker fusion (CFF) test that measures a sensitivity threshold to provide information about the temporal responsiveness of visual pathways, etc. These assessments are known to one skilled in the art.
The following examples provide further embodiments of the invention.
LASIK surgery was performed on each eye of twenty-two patients. Each patient was enrolled in a single-center clinical trial.
In this surgery, corneal nerves in the hinge region remained unaffected (i.e., they remained both present and intact). Corneal nerves in the remainder of the flap, however, were mechanically dissected during surgery to mobilize the flap, but remained structurally present (i.e., they remained present but not intact).
It is known that after this type of surgery, nerves that have been severed gradually and over the course of months reenervate the cornea. Corneal sensation is thus gradually restored at a slow rate during normal post-surgical recovery.
The rate of corneal sensation restoration by a commercially available 0.05% cyclosporine emulsion applied to an eye after LASIK surgery was assessed. Twenty-two patients underwent LASIK surgery using a combined Excimer laser with a lamellar corneal flap technique, as known to one skilled the art and as discussed in, e.g., Linna et al., Exp. Eye Res. (1998) 66, 755, which is expressly incorporated by reference herein. One eye was randomly assigned as a "study" eye, and the other eye was the "control" eye. The surgeon was instructed to ensure that the hinge position of the LASIK flap was the same for both eyes of the patient, because this affected corneal sensation in that the loss of corneal sensation was known to be greater in eyes with a superior-hinge flap than in eyes with a nasal-hinge flap.
At the completion of the LASIK surgery, one drop of cyclosporine 0.05% ophthalmic emulsion was topically applied to one eye ("study eye") of the patient. After at least ten minutes, one drop of each of prednisolone acetate (ECONOPRED® Alcon, Ft. Worth Tex.) and moxifloxacin (VIGAMOX®. Alcon) were topically applied to the same "study" eye. One drop of each of prednisolone acetate and moxifloxacin were topically applied to the "control" eye of the same patient. Cyclosporine was not applied to the "control" eye.
Patients were instructed to administer the following medicaments to the "study" eye, starting on the day of surgery: one drop of cyclosporine 0.05% at about twelve hour intervals, and one drop of each of prednisolone acetate and moxifloxacin four times a day, waiting at least ten minutes after cyclosporine administration. Patients were instructed to administer the following medicaments to the "control" eye, starting on the day of surgery: one drop of each of prednisolone acetate and moxifloxacin four times a day,
The cyclosporine administration regimen to the "study" eye continued until the three month post-surgical evaluation. The prednisolone acetate and moxifloxacin regimen administration to the "study" eye continued until a one-week post-surgical evaluation, then changed to twice a day administration for two days, then once a day for two days. An ocular lubricant (SYSTANE® Alcon) was administered to both "study" and "control" eyes once on the day of surgery, and thereafter as needed.
Patients were evaluated for corneal sensitivity pre-surgery and immediately post-surgery, then at one- and three-months post-surgery. Nineteen patients (thirty-eight eyes) were evaluated at the three-month examination; the remaining patients did not report. Post-surgical corneal sensitivity recovery of the 19 study eyes and 19 control eyes was compared.
Corneal sensitivity was measured using the Cochet-Bonnet esthesiometer in accord with the manufacturer's instructions. The instrument is a nylon filament 60.0 mm long and 0.12 mm diameter. The force exerted by the filament when it touches the cornea is inversely proportional to its length. The same experienced examiner made all measurements whenever possible.
Measurements were obtained at nine different areas of the cornea, as diagrammed in
For post-surgical examination, the examiner was unaware of which patient eye was the "study" eye and which eye was the "control" eye. The patient, in a supine position looking straight ahead, was asked to indicate to the examiner when the stimulus was felt (positive response). The filament was moved toward the cornea smoothly at a perpendicular angle, guided by its corneal reflex. Contact was detected by a slight bending in the filament. If the patient did not respond to the first contact, the length of the filament was decreased by 5.0 mm, and the procedure was repeated until the patient reported feeling corneal contact. The longest filament length at which a minimum of three stimulus applications produced a positive response from the patient was recorded. This was considered the corneal touch threshold.
Data were statistically evaluated using the Wilcoxon Sum Rank Test. Statistical significance of differences in corneal sensitivity was determined by comparing the two eyes of each patient (one study eye and one control eye in each patient), using statistical significance at p =0.05.
Data for each of the nine areas of the eye in which corneal sensitivity was assessed in each of the 19 control and 19 study eyes are shown in
Corneal sensitivity at each of the four peripheral quadrants of untreated cornea, shown in
Patients were also evaluated by slit lamp examination. The eyelid margin, conjunctiva, cornea, and anterior segment were thoroughly examined. For each examination at all time points (pre-surgery and post-surgery one day, one week, one month, and three months) there were no statistically significant differences between the "study" and "control" eyes.
Patient subjective responses (pre-surgery and post-surgery one week, one month, and three months) to a questionnaire were evaluated. For each eye, patients were asked to rate (never, rarely, sometimes, often, all of the time) sensation (dry, sticky, gritty, sore, watery, burning, "foreign body"); vision (blurred); appearance (red, crusting or discharge on lashes); and sensitivity to light and wind/air conditioning. At all time points, there were no statistically significant differences between the "study" and "control" eyes with the exception of "foreign body sensation". This was more noticeable in the "study" eye one week post-surgery (p=0.047), although at one and three months post-surgery, there was no significant difference between the "study" and "control" eyes (p=0.102 and p=0.317, respectively).
These data demonstrated efficacy of topically applied cyclosporine 0.05% ophthalmic solution in significantly improved corneal sensitivity within three months post-LASIK. This provided strong evidence that cyclosporine 0.05% ophthalmic solution promoted corneal nerve recovery.
Photorefractive keratectomy (PRK) surgery is performed on at least one eye of a patient. In PRK, the corneal surface is reshaped using an Excimer laser for surface ablation and removal of the epithelium and stroma; it can also be performed under an epithelial flap. The protective superficial layers of the cornea heal post-operatively. Patients who have undergone PRK will typically require more time than patients who have undergone LASIK to achieve their best vision and restore corneal sensitivity. Outcomes of PRK and LASIK are comparable at the sixth postsurgical month. There may be a greater risk of scarring (haze) and unpredictable healing of the cornea with PRK. The risk of infection is also slightly higher with PRK than with LASIK, although infections following either procedure are rare.
After surgery, 0.05% cyclosporine is provided to the affected eye. Dosing intervals, assessment techniques, etc. are the same as in Example 1. Enhanced recovery rate of corneal sensitivity is provided.
Laser thermal keratoplasty (LTK) is performed on at least one eye of a patient. Energy from a holmium-YAG laser is applied in a predetermined pattern to shrink collagen fibers and thus steepen the cornea. After the procedure, 0.05% cyclosporine is provided to the affected eye. Dosing intervals, assessment techniques, etc. are the same as in Example 1. Enhanced recovery rate of corneal sensitivity is provided.
Conductive keratoplasty (CK) is performed on at least one eye of a patient. Low-level radiofrequency (RF) energy is administered through a fine probe. Using the probe, RF energy is applied to specific sites in a pre-set marked circular pattern (e.g., using a dye) on the outer cornea to shrink or ablate small areas of corneal tissue. The circular shrinkage pattern creates a constrictive band, thus increasing the overall curvature of the cornea.
After the procedure, 0.05% cyclosporine is provided to the affected eye. Dosing intervals, assessment techniques, etc. are the same as in Example 1. Enhanced recovery rate of corneal sensitivity is provided.
Laser-assisted in situ epithelial keratomileusis (LASEK) is performed on at least one eye of a patient. After surgery, 0.05% cyclosporine is provided to the affected eye. Dosing intervals, assessment techniques, etc. are the same as in Example 1. Enhanced recovery rate of corneal sensitivity is provided. Other variations or embodiments of the invention will also be apparent to one of ordinary skill in the art from the above description. As one example, the invention may be used to facilitate growth of transplanted neuronal cells, either mature or immature, and/or stem cells in the eye or brain. As another example, other ocular routes of administration and injection sites and forms are also contemplated. As another example, the invention may be used in patients who have experienced ocular trauma, ischemia, inflammation, etc. Thus, the forgoing embodiments are not to be construed as limiting the scope of this invention.
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An assistive device is a tool or implement that makes a particular function easier or possible to perform. An assistive device may be as simple as an electric toothbrush, or as elaborate as an environmental control system that can be operated with a mouth switch by persons who have lost the use of their limbs.
Assistive devices can help persons with disabilities perform many activities of daily living.
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Identification of 3′ gene ends using transcriptional and genomic conservation across vertebrates
Author affiliations
1 International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, I 34149, Trieste, Italy
2 Present address: EMBL Monterotondo Adriano Buzzati-Traverso Campus, Via Ramarini 32, 00015, Monterotondo, Italy
Citation and License
BMC Genomics 2012, 13:708 doi:10.1186/1471-2164-13-708Published: 18 December 2012
In higher eukaryotes, gene expression is regulated at different levels. In particular, 3′UTRs play a central role in translation, stability and subcellular localization of transcripts. In recent years, the development of high throughput sequencing techniques has facilitated the acquisition of transcriptional data at a genome wide level. However, annotation of the 3′ ends of genes is still incomplete, thus limiting the interpretation of the data generated. For example, we have previously reported two different genes, ADD2 and CPEB3, with conserved 3′UTR alternative isoforms not annotated in the current versions of Ensembl and RefSeq human databases.
In order to evaluate the existence of other conserved 3′ ends not annotated in these databases we have now used comparative genomics and transcriptomics across several vertebrate species. In general, we have observed that 3′UTR conservation is lost after the end of the mature transcript. Using this change in conservation before and after the 3′ end of the mature transcripts we have shown that many conserved ends were still not annotated. In addition, we used orthologous transcripts to predict 3′UTR extensions and validated these predictions using total RNA sequencing data. Finally, we used this method to identify not annotated 3′ ends in rats and dogs. As a result, we report several hundred novel 3′UTR extensions in rats and a few thousand in dogs.
The methods presented here can efficiently facilitate the identification of not-yet-annotated conserved 3′UTR extensions. The application of these methods will increase the confidence of orthologous gene models across vertebrates.


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33 year old man | Euless, TX, USA | Seeking women 18-35 within 50 miles
Why not? Lets see where this goes.
Enjoy the simple things, spending time with friends and family , dinner, football , traveling... or just a nice relaxing Sunday at home - a movie and some great food. Family is very important. Looking for someone that can be a great friend and is seeking a relationship based on trust and communication. Not that i'm looking to get married tomorrow, but definitely looking for a serious relationship. I am a very hard working and genuine guy that learned to appreciate life and would love to find someone to share it with.
Coffee and conversation , Cooking , Fishing/Hunting , Movies/Videos , Music and concerts , Playing sports , Travel/Sightseeing , Watching sports , Wine tasting
Auto racing / Motorcross, Baseball, Billiards / Pool, Football, Golf, Tennis / Racquet sports, VolleyBall
I exercise 3-4 times per week.
I like Birds, Cats, Dogs, Fish
I would love to have a Dog and maybe a Cat, But i don't have a yard right now for them to play and also travel for work so its just not fair to them at this time. But have had Dogs more most of my youth.
Some other viewpoint
Culebra Island off the coast of Puerto Rico A Parador in Rincon Rio de Janeiro Brazil
I really like all types of music, from Pop to Reggae and everything in between. Love all kinds of food also, Italian, love criollo food, and cant forget a great steak :) .
golf, movies, Traveling , Football games
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1986 Rusty Staub Original Painting by LeRoy Neiman....
2014 February 22 - 23 Sports Collectibles Platinum Night Auction - New York #7100
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Ukrainian Institute of America at The Fletcher-Sinclair Mansion
2 East 79th Street
New York, NY 10075
|Printable auction results for all items in the 2014 February 22 - 23 Sports Collectibles Platinum Night Auction - New York.
A masterful portrait of "Le Grand Orange."1986 Rusty Staub Original Painting by LeRoy Neiman. Many fans assign credit for the Mets' second National League Championship in 1973 to the thunderous bat of their star right-fielder, who blasted three homers and drove in five runs before the spectacular robbery of an extra-base hit off the bat of Reds slugger Dan Driessen separated Staub's shoulder and limited his effectiveness in the subsequent World Series battle with the Oakland A's.
His productive bat (Staub's 105 RBI's in 1975 stood as a Mets record for over a decade) and nifty fielding endured him to the fans at Shea during nine seasons with the club. The style is classic Neiman, thickly applied paint in kaleidoscopic bursts of color, the palette here relying heavily on the Mets blue and orange. The work measures 19x25" in size and is signed and dated "'86" by Neiman at lower left. On reverse, Neiman's distinctive script titles the work, "Grand Orange Grand Slam."
Though Neiman has long served as the most recognizable American artist of the post-war era, his recent passing has added rocket fuel to the fire of his market, with four of his works soaring into six-figure territory in recent Heritage auctions. We expect the climb in pricing to continue, and for smart investors to be paying close attention when this classic work hits the block.
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Shane Segovia had an RBI single to pull the Tigers within one run in the fourth.
April 14, 2013
NASHVILLE, Tenn. - The Mizzou baseball team was defeated by No. 3 Vanderbilt, 16-4, on Sunday afternoon (April 14) at Hawkins Field as Vanderbilt completed the three-game sweep of the Tigers. In the series, Vanderbilt outscored Mizzou 39-10 and pounded out 54 hits in the three-game sweep. Reigning SEC Player of the Week Keaton Steele (St. Joseph, Mo.) was tagged with his second loss of the season after allowing nine runs (eight earned) over 3.2 innings while walking four and striking out three. Steele was the first Tiger to start a game on the mound and hit in the three-hole of the lineup since on May 12, 2001, in an 8-5 win over Texas A&M.
Mizzou allowed 16 runs on 18 hits on Sunday while it drops to 12-20 on the season and 4-11 in SEC play. The third-ranked Commodores move to 32-4 on the season and 14-1 in SEC play while they have won 14 in a row. Mizzou pulled as close as 4-3 in the top of the fourth inning, but Vanderbilt answered with six runs in the bottom of that frame and then added five more over the sixth and the seventh innings to pull away. Lefty Philip Pfeifer earned his third win of the season for Vanderbilt, going 4.0 innings, allowing three runs (two earned) on four hits while walking three and striking out two.
The theme of the weekend up until Sunday was the ability of Vanderbilt to get infield hits and that's how the Commodores began the game again with a bunt single by Tony Kemp. Steele then allowed a double to two-hole hitter Xavier Turner and an RBI fielder's choice game the Commodores an early 1-0 lead. Connor Harrell then singled back up the middle past the drawn-in infield to plate another run as the Commodores plated three in the first. Mizzou got its first two men aboard in the second on walks, but a diving catch at second by Kemp robbed Kendall Keeton (Columbia, Tenn.) of an RBI single and then Josh Lester (Columbus, Ga.) was picked off at first before Logan Pearson (Bentonville, Ark.) flew out to left to end the threat.
Vanderbilt then got another run after a pair of tough errors and a single by Harrell, his second RBI knock of the day, to take a 4-0 lead through two innings. After a scoreless third inning, Mizzou finally got to Vandy starter Phillip Pfeifer, tallying four-straight two-out hits to score three runs. With runners on the corners, Pearson singled through the right side to plate Lester and the big hit came off the bat of Shane Segovia (Sinton, Texas), who laced a 1-2 pitch into right field to score Keeton and the throw from right field got away, allowing Pearson to score, pulling the Tigers within one at 4-3.
But Kemp struck again as he used his wheels to leg out a leadoff triple on a ball hit into right center and he scored on a fielder's choice, again off the bat of Harrell. Segovia then committed a throwing error as he tried to get the out at second, allowing the runner to go to third and that runner scored on another fielder's choice, giving Vanderbilt a 6-3 lead. Another single scored a run before Spencer Navin hit a two-run bomb to left center to give the Commodores a 9-3 lead. They added another run as they sent 11 batters to the plate and scored six in the inning to take an 10-3 lead into the fifth.
VU then scored two more in the sixth. Mizzou got a run in the seventh on a Dane Opel (Edwardsville, Ill.) single as he tallied a double to right center to score Case Munson (St. Charles, Mo.) from first. The RBI was Opel's team-leading 18th this season, tying him with Dylan Kelly (Roswell, Ga.) but just as Vandy had done all weekend, it answered with three more in the bottom half of the seventh to take a 15-4 lead.
Mizzou will close its home-and-home series with in-state rival Missouri State on Wednesday (April 17) at 6 p.m. It will be the Tigers' first home game in the month of April after playing the last eight games away from Taylor Stadium. For all the latest on Mizzou baseball, stay tuned to MUTigers.com and follow the team on twitter @MUTigerBaseball.
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Revenge Season 3, Episode 14 Spoilers Roundup: Everything You Need to Know About "Payback"
The wait for the next episode of Revenge, Season 3, Episode 14 ("Payback") is almost over! The episode airs this Sunday, March 9. Is two days too long to wait? Well, at least we have plenty of spoilers to tide us over until the episode finally gets here.
So what can we expect when we finally take a trip back to the Hamptons? We've gathered all of the biggest spoilers and hints about the episode right here.
Read spoilers at your own risk! For more on Wetpaint Entertainment's philosophy on spoilers, click here.
Emily's blackouts threaten everything. It sounds like Emily's blackout-related problems are just beginning because the official synopsis of the episode teases, "Emily's mysterious blackouts threaten everything and everyone she holds dear." It's clear how the blackouts threaten her plans (it's hard to be a manipulative mastermind if you keep forgetting what you've done), but the part where they threaten "everyone she holds dear" has us worried. Could Aiden, Nolan, Jack, or Charlotte find themselves in trouble because of something Em does?
Ghosts from Victoria's past. Meanwhile, the synopsis teases that "Victoria confronts two formidable ghosts from her past."
Who are those ghosts? We're thinking one is the first Ms. Grayson, who we saw at the very end of Episode 13. We know she's going to face off against Victoria in this episode, which should be fantastic. Anyone who would marry Conrad must know how to fight dirty.
The second is probably Patrick's father, who is featured heavily in the promo pics for the episode.
Emily vs. Niko. Meanwhile, thanks to the promos of the episode, we know there's an Emily vs. Niko fight, which should be fun.
Catch this episode of Revenge on Sunday, March 9 at 10 p.m. ET on ABC.


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Hi Clayton,Originally Posted by claytume
your print arrived while I was in Boston last week. It's fantastic! I'm not surprised you sent it packaged the way it was, not much else would make sense. Certainly one of the most unusual prints I've received so far in these exchanges. Thanks a lot. Mine should be out by end of this week before I head to Singapore on Sunday.


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Politics change, but Torah is immutable. It will withstand all political changes and something that is not acceptable by Torah will always be unacceptable no matter how "politically incorrect" it will become in the eyes of those who don't subscribe to Torah values.
If selected for publication, it will appear as soon as possible on Haaretz.com.


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GeForce 5900 FX PPL post ya scores
I just got my new 5900 and I',m experiencing some really bad frame rate in 3Dmark03. My overall score was 4095. Please post your scores so I can compare.
Re: GeForce 5900 FX PPL post ya scores
Omega drivers. Ew. And, the 3DMark optimizations in 44.03 are disabled by version 330. So, in reality, that's about the score you should get, not this 6700 crap.
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The New York Times ran a good front-page story yesterday that detailed the racism of John Tanton, the architect of the modern anti-immigration movement, something we have been writing about since 2002 (here, here and here). But the Times' story gave the organizations Tanton founded, including the Federation for American Immigration Reform (FAIR), a bit of a pass, implying that charges of racism against them turned almost entirely on their association with Tanton. The truth of the matter is that, particularly in the case of FAIR, the organizational apple has not fallen far from the founder's tree.
Dan Stein, FAIR's president, has his own notorious history of racism. He has repeatedly attacked the Immigration and Nationality Act of 1965 – enacted, in President Lyndon B. Johnson's words, to end the "harsh injustice" of our country's then-racist immigration quota system – as a disaster for Western civilization and Anglo-Saxon dominance. In a 1994 oral history, Stein told his interviewer, Tanton, that those who supported the 1965 reform wanted to "retaliate against Anglo-Saxon dominance" and that this "revengism" against whites had created a policy that is causing "chaos and will continue to create chaos."
In a 1991 memo entitled "The Defenders of American Culture Rise to the Call to Arms," Stein said he hoped that mounting criticism of multiculturalism would eventually lead to attacks on the 1965 Act, which he called "a key mistake in national policy" and a "source of error."
Like Tanton, Stein takes a dim view of today's immigrants. He has warned that immigrants are engaged in "competitive breeding" aimed at diminishing white power and that "[m]any of them hate America, hate everything the United States stands for." Stein led FAIR's efforts to win funding from the racist Pioneer Fund, which promotes the idea that whites are genetically superior, saying in 1993 that his "job [was] to get every dime of Pioneer's money." Bizarrely, FAIR describes the fund on its website as supporting "equal opportunity for all Americans."
Stein also served as editorial adviser to The Social Contract, a nativist hate journal published by Tanton, when it ran a particularly virulent special issue that was entitled, "Europhobia: The Hostility Toward European-Descended Americans." The lead article argued that multiculturalism was replacing "successful Euro-American culture" with "dysfunctional Third World cultures."
Other FAIR leaders have participated in racist groups. Rick Oltman, FAIR's longtime western regional coordinator, spoke as part of a 1997 immigration panel put on by the Council of Conservative Citizens (CCC), a hate group that has described black people as a "retrograde species of humanity." CCC publications at the time listed Oltman as a member. The FAIR official who followed Oltman in his position, Joseph Turner, was on record before joining FAIR as saying that being a white separatist did not imply a person was racist. Jim Staudenraus, FAIR's eastern regional coordinator, participated in an anti-immigration conference in 2002 with Jared Taylor, a prominent white nationalist and friend of John Tanton. In 2007, a senior FAIR official met with leaders of Vlaams Belang, a Belgian political party that officials in that country outlawed in a previous incarnation (Vlaams Blok) as a "criminal organization" because of its racist anti-immigrant views.
FAIR's board also has extremist connections. FAIR board member Donald Collins writes frequently for VDARE.com, a nativist website named after Virginia Dare, said to have been the first English child born in the New World. (VDARE is dedicated to bashing immigrants and has published the work of many white nationalists and anti-Semites.) Collins' articles have focused on attacking the Catholic Church for its liberal stance on immigration. One accused Los Angeles Archbishop Roger Mahony of selling out his country "in exchange for more temporal power and glory." Another claimed bishops were "infiltrating and manipulating the American political process" to dismantle the separation of church and state — the classic calumny directed at American Catholics for decades by the Klan and others. Another person linked to VDARE is Joe Guzzardi, a member of FAIR's board of advisors who has worked as an editor of the site. Other members of FAIR's board of directors have offered similar sentiments. Former Colorado Gov. Richard Lamm once said that "new cultures" in America were "diluting what we are and who we are."
Although Stein tried to imply otherwise in his interview with the Times, Tanton remains a force at FAIR, where he is on the board of directors. In its 2004 annual report, FAIR praised Tanton for "visionary qualities that have not waned one bit." In 2009, FAIR President Dan Stein told the Washington Post that Tanton is a "Renaissance man" of wide-ranging "intellect." Tanton is also on the board of Pro English and continues to publish the Social Contract.
For more details on FAIR's extremism, which includes racist anti-immigrant television programming and the dissemination of anti-immigrant propaganda, please read here.
Given FAIR's views of immigrants, it is not surprising that FAIR's latest efforts have been to pass punishing anti-immigrant legislation at the local and state levels. At the local level, these efforts have led to racial strife and bankrupted communities. The SPLC has documented these effects in our report "When Mr. Kobach Comes to Town: Nativist Laws and the Communities They Damage." Kris Kobach is a lawyer for FAIR's legal arm, the Immigration Reform Law Institute.
At the state level, FAIR is a partner to State Legislators for Legal Immigration (SLLI), which hopes to pass anti-immigrant laws. The group, founded in 2007 and filled with legislators holding extremist views, is rabidly anti-immigrant. It attributes to "illegal aliens" what it describes as "[i]ncreasingly documented incidences of homicide, identity theft, property theft, serious infectious diseases, drug running, gang violence, human trafficking, terrorism and growing cost to taxpayers." Its founder, Republican Pennsylvania state Rep. Daryl Metcalfe, writes on SLLI's website that "the personal and economic safety" of all Americans is threatened by "the ongoing invasion of illegal aliens" and compares the situation to that facing the settlers during the American Revolution. SLLI and FAIR have this year devoted themselves to gutting the 14th Amendment's promise of birthright citizenship. The amendment was approved in the aftermath of the Civil War to guarantee citizenship to all persons born in this country. FAIR and its allies are willing to undermine that crucial part of the Constitution to further their anti-immigrant agenda.
For more information about other Tanton-created organizations with anti-immigrant track records mentioned in the Times' story, please read our profiles of NumbersUSA and the Center for Immigration Studies.


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On 14 December 2012, José Manuel Barroso, President of the EC, and Herman van Rompuy, President of the European Council, gave a press conference following the first evening of the European Council, held on 13-14 December 2012 in Brussels.
On this occasion, they underlined the good discussion on the future of the Economic and Monetary Union. José Manuel Barroso also welcomed the endorsement of agreements on the Single Supervisory Mechanism and on Greece.


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Literature Retold as Bite-Size Gangsta Rap Entertains and, Often, Educates ; Thug Notes Site Follows Trend of Applying Street Sensibility to High Culture
Genzlinger, Neil, International New York Times
Thug Notes, a site that reduces works of literature to their hip- hop essence, is a deliciously executed example of a trend that has been around for years: the application of street sensibility to high culture.
In 1848, a reviewer for Graham's Magazine described "Wuthering Heights" as "a compound of vulgar depravity and unnatural horrors, such as we might suppose a person, inspired by a mixture of brandy and gunpowder, might write for the edification of fifth-rate blackguards." Presumably, this grumpy writer would have cared even less for the Thug Notes version.
That Emily Bronte novel is among the latest subjects tackled on Thug-Notes.com, a website where fine literature is reduced to its hip-hop essence. A genial fellow using the moniker Sparky Sweets, Ph.D. serves up video summaries of classics in the language of the street, throwing in a minute or two of analysis for good measure. Dr. Sweets, a black man whose wardrobe leans toward shorts, tank tops and assorted do-rags and caps, sits in a somber-looking library worthy of the Public Broadcasting Service and holds forth about a new volume each week. The site's motto: "Classical Literature. Original Gangster."
In the world of Dr. Sweets (who is actually a comedian named Greg Edwards), Queequeg from "Moby-Dick" is "some tatted-up harpooner." Jay Gatsby is "a rich playboy with that mad Mitt Romney money." And the characters of a beloved Shakespeare play include "Romeo's homieos, Benvolio and Mercutio."
SparkNotes and others, of course, have been summarizing the classics for years, but their cheat sheets have merely made literature's dusty volumes drastically shorter, not less boring for the lazy and unappreciative. Mr. Edwards, speaking in character as Dr. Sweets in an interview with The Tampa Bay Times last fall, described Thug Notes as "my way of trivializing academia's attempt at making literature exclusionary by showing that even highbrow academic concepts can be communicated in a clear and open fashion."
The site manages to turn "Wuthering Heights," "Pride and Prejudice" and other tomes into bite-size fun (the videos are generally under five minutes) while conveying a certain respect toward the source material.
The good doctor's summary of "Romeo and Juliet" may be full of unpublishable slang, but it ends with a discussion of the clashes of opposites in the work and whether it can rightly be labeled a tragedy. He may dismiss the core of "Moby-Dick" as "about 500 pages of Ishmael going off about whaling" (finishing that phrase with an expletive), but he has thesis-worthy thoughts about the symbolism of the whale, of the quest and of the ship -- the Rachel -- that rescues Ishmael.
"Keep floating, homies," he concludes, "'cause somewhere out there, we all got our own Rachel that's there to save us."
Thug Notes is a deliciously executed example of a trend that has been around for years: the application of street sensibility to high- culture, high-concept areas and, more generally, any place where it is not expected. …


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BLOOMINGTON, Ind. -- A team of biologists from Indiana University and Brown University believes it has discovered the mechanism by which interacting mutations in mitochondrial and nuclear DNA produce an incompatible genotype that reduces reproductive fitness and delays development in fruit flies.
The new research, led by IU biologists Kristi Montooth and Colin Meiklejohn and including former IU undergraduate researcher Mo Siddiq, describes the cause and consequences of an interaction between the two genomes that co-exist within eukaryotic cells. Animal mitochondrial DNA, or mtDNA, is a small but important genome that encodes a handful of proteins that are essential to oxidative phosphorylation, the pathway that produces the adenosine triphosphate molecule that fuels cellular metabolism.
With this new characterization of a disruptive interaction between mtDNA and nuclear DNA mutations, the scientists provide one of the few mapped cases of a fitness-reducing mitochondrial-nuclear incompatibility.
The genetic interaction that IU biologists mapped, in collaboration with Brown University biologist David Rand, is between mutations that are present in natural populations, rather than being induced in the lab. This has important consequences for understanding genetically complex human diseases.
Many human diseases, such as neuromuscular and neurodegenerative disorders, are associated with mutations in mitochondrial transfer RNAs, or tRNAs, but a single mutation can be highly variable in the degree to which it leads to disease.
Montooth and her colleagues' findings suggest that the combined mitochondrial-nuclear genotype for tRNAs and their tRNA synthetases may, in fact, be a better predictor of disease.
"Interactions between mitochondrial and nuclear DNA for fitness have been documented in many organisms, but rarely has the genetic or mechanistic basis of these interactions been elucidated," said Montooth,
|Contact: Steve Chaplin
Indiana University