HK1200365B - Liquid formulations for treatment of diseases or conditions - Google Patents

Description

Liquid preparations for treating diseases or conditions

The present invention is a divisional application of the invention patent with application number 200680008018.X, application date February 9, 2006, and invention name being liquid preparation for treating diseases or conditions.

Technical Field

Described herein are liquid formulations for treating, preventing, inhibiting, delaying the onset of, or regressing a disease or condition by delivering a therapeutic agent to a subject (including but not limited to a human subject), including but not limited to treating age-related macular degeneration (AMD) by delivering a liquid formulation comprising a therapeutic agent to the eye of a subject (including but not limited to a human subject). Non-limiting examples of the liquid formulations include solutions, suspensions, and in situ gelling formulations.

Cross-reference with related applications

This application is related to and claims priority to U.S. Provisional Patent Application Serial No. 60/664,040, entitled "Liquid Formulations For Treatment Of Diseases Or Conditions," filed on March 21, 2005, U.S. Provisional Patent Application Serial No. 60/664,306, entitled "In Situ Gelling Formulations And Liquid Formulations For Treatment of Diseases Or Conditions," filed on March 21, 2005, and U.S. Provisional Patent Application Serial No. 60/651,790, entitled "Formulations For Ocular Treatment," filed on February 9, 2005, each of which is incorporated herein by reference in its entirety for all purposes.

Technical Background

The retina of the eye contains light-sensitive cones and rods. At the center of the retina is the macula, which is about 1/3 to 1/2 cm in diameter. The macula provides detailed vision, especially in the center (fovea), because cones are denser. Blood vessels, ganglion cells, the inner nuclear layer and cells, and the plexiform layer have all moved to one side (rather than resting on the cones), allowing light to take a more direct path to the cones.

Under the retina lies the choroid, which includes a network of blood vessels embedded in fibrous tissue and a dark pigmented epithelium covering the choroidal layer. The choroidal blood vessels provide nutrition to the retina, particularly its photoreceptors.

There are many retinal diseases for which there is currently no treatment or for which current treatments are suboptimal. Retinal diseases such as uveitis (inflammation of the uveum: sclera, ciliary body, and choroid), central retinal vein occlusive disease (CRVO), branch retinal vein occlusion (BRVO), macular degeneration, macular edema, proliferative diabetic retinopathy, and retinal detachment are generally all retinal diseases that are difficult to treat with conventional therapies.

Age-related macular degeneration (AMD) is the leading cause of severe vision loss in individuals older than 60 years in the United States. AMD occurs in either an atrophic or, less commonly, an exudative form. The atrophic form of AMD is also known as "dry AMD," while the exudative form is also known as "wet AMD."

In exudative AMD, blood vessel grows through the defect in Bruch's membrane (being below retinal pigment epithelium in some cases) from the choriocapillaris layer.The tissue of serous or hemorrhagic exudate that overflows from these vessels causes macular fibrovascular scarring and the incidental degeneration, retinal pigment epithelium detachment and tearing, vitreous hemorrhage and central vision permanent loss of the optic nerve retina.This process occupies more than 80% of the significant vision loss case in the experimenter suffering from AMD.Current or impending treatment comprises laser photocoagulation, photodynamic therapy, with VEGF antibody fragment treatment, with PEGylated aptamer treatment and with some small molecule agent treatment.

Some studies have recently described the purposes (Macular Photocoagulation Study Groups (1991) in Arch.Ophthal.109:1220 in the treatment of primary or recurrent neovascular lesions relevant to AMD by laser photocoagulation; Arch.Ophthal109:1232; Arch.Ophthal.109:1242). Unfortunately, the AMD patient with subfoveal damage who underwent laser treatment experienced a very rapid decrease in visual acuity (average 3 lines) during a 3-month follow-up observation. In addition, after treatment for 2 years, the treated eyes only had a small amount of visual improvement (average 20/320 and 20/400 respectively) compared to their untreated eyes. Another shortcoming of the method is that postoperative vision deteriorates immediately.

Photodynamic therapy (PDT) is a type of phototherapy—a category encompassing all treatments that use light to produce a beneficial response in a subject. Ideally, PDT destroys unwanted tissue while sparing healthy tissue. A compound called a photosensitizer is typically administered to the subject. The photosensitizer itself typically has little or no effect on the subject. When light (usually from a laser) is directed at tissue containing the photosensitizer, the agent becomes activated and begins destroying the target tissue. Because the light directed at the subject is confined to a specific target area, PDT can be used to selectively target abnormal tissue, sparing surrounding healthy tissue. PDT is currently used to treat retinal diseases such as AMD. PDT is currently the mainstay of treatment for subfoveal choroidal neovascularization in patients with AMD (Photodynamic Therapy for Subfoveal Choroidal Neovascularization in Age-Related Macular Degeneration with Verteporfin (TAP Research Group) Arch Ophthalmol. 1999 117:1329-1345).

Choroidal neovascularization (CNV) has been shown to be resistant to treatment in most cases. Conventional laser therapy can remove CNV and help preserve vision without involving the center of the retina, but this is limited to about 10% of cases. Unfortunately, even after successful conventional laser photocoagulation, neovascularization recurs in 50%-70% of eyes (50% at 3 years and >60% at 5 years). (Macular Photocoagulation Study Group, Arch. Ophthalmol. 204: 694-701 (1986)). In addition, many subjects with CNV are not good candidates for laser therapy because the CNV is too large for laser treatment or the location cannot be determined so that the doctor cannot accurately aim the laser. Although photodynamic therapy is used in up to 50% of new cases of subfoveal CNV, it has only a small benefit on the natural history and usually delays the progression of vision loss rather than improving vision that has already decreased after subfoveal damage. PDT is not preventive and is not definitive. Each subject typically requires several PDT treatments, and certain subtypes of CNV do not progress as well as others.

Thus, there remains a long felt need for methods, compositions, and formulations that can be used to optimally prevent or significantly inhibit choroidal neovascularization and for preventing and treating wet AMD.

In addition to AMD, choroidal neovascularization has been associated with such retinal diseases as presumed ocular histoplasmosis, myopic degeneration, angioid streaks, idiopathic central serous chorioretinopathy, inflammatory diseases of the retina and/or choroid, and ocular trauma. Angiogenic impairment associated with neovascularization occurs in a variety of diseases, including diabetic retinopathy, venous occlusions, sickle cell retinopathy, retinopathy of prematurity, retinal detachment, ocular ischemia, and trauma.

Uveitis is another retinal condition that has proven difficult to treat using existing therapies. Uveitis is a general term used to designate inflammation of any component of the uveal tract. The uveal tract of the eye is composed of the sclera, ciliary body, and choroid. Inflammation of the overlying retina (called retinitis) or inflammation of the optic nerve (called optic neuritis) can occur with or without uveitis.

Uveitis is most commonly classified anatomically as anterior, middle, posterior, or diffuse. Posterior uveitis refers to any of various forms of retinitis, choroiditis, or optic neuritis. Diffused uveitis refers to inflammation involving all parts of the eye, including the anterior, middle, and posterior structures.

Symptoms and signs of uveitis can be mild and vary widely depending on the location and severity of the inflammation. For posterior uveitis, the most common symptoms include the presence of suspended matter and decreased vision. Subjects with posterior uveitis may also have cells in the vitreous humor, white or yellow-white lesions in the retina and/or underlying choroid, exudative retinal detachment, retinal vasculitis, and optic nerve edema.

Ophthalmic complications of uveitis can result in significant and irreversible vision loss, particularly when unrecognized or inadequately treated. The most common complications of posterior uveitis include retinal detachment, neovascularization of the retina, optic nerve, or sclera, and cystoid macular edema.

Macular edema (ME) occurs when swelling, leakage, and hard exudates are documented in the macula (the central 5% of the retina most critical for vision) in background diabetic retinopathy (BDR). Background diabetic retinopathy (BDR) is typically composed of small retinal aneurysms, which are caused by alterations in the retinal microcirculation. These small aneurysms are often the earliest visible changes in retinopathy seen with ophthalmoscopy, appearing as scattered red spots in the retina where tiny, weakened blood vessels have bulged. Over the course of background diabetic retinopathy, visual findings progress to include cotton-wool spots, intraretinal hemorrhages, fluid leaking from the retinal capillaries, and retinal exudates. Increased vascular permeability is also associated with elevated levels of local growth factors, such as vascular endothelial growth factor. The macula is rich in cones, the nerve endings that are responsible for color perception and daytime vision. When increased retinal capillary permeability affects the macula, the central field of vision, or just the edges of the central field of vision, becomes blurred, like looking through cellophane. Vision loss can develop over a period of months and can be very disturbing due to the inability to focus clearly. ME is a common cause of several vision impairments.

There have been attempts to use drugs to treat CNV and its related diseases and conditions, as well as other conditions such as macular edema and chronic inflammation. For example, the use of rapamycin to inhibit CNV and wet AMD is described in U.S. Application No. 10/665,203, which is incorporated herein by reference in its entirety. The use of rapamycin to treat inflammatory diseases of the eye is described in U.S. Patent No. 5,387,589, entitled "Method of Treating Ocular Inflammation," inventor Prassad Kulkarni, assigned to the University of Louisville Research Foundation, the contents of which are incorporated herein by reference in their entirety.

Particularly for chronic diseases, including those described herein, there is a great need for long-acting methods for delivering therapeutic agents to the eye (e.g., to the posterior segment to treat CNV in diseases such as AMD, macular edema, proliferative retinopathy, and chronic inflammation. Formulations that provide extended delivery of therapeutic agents are more comfortable and convenient for the subject because the frequency of ocular injections of the therapeutic agent is reduced.

Delivering therapeutic agents directly to the eye rather than systemically can be advantageous because the concentration of the therapeutic agent at the site of action is increased relative to the concentration of the therapeutic agent in the subject's circulation. Additionally, systemic delivery of therapeutic agents to treat posterior segment diseases can have undesirable side effects. Therefore, localized drug delivery can improve efficiency while reducing side effects and systemic toxicity.

SUMMARY OF THE INVENTION

The methods, compositions, and liquid formulations described herein allow for the delivery of therapeutic agents to a subject (including, but not limited to, a human subject) or to the subject's eye. Described herein are methods, compositions, and liquid formulations for the extended delivery of a variety of therapeutic agents useful for treating, preventing, inhibiting, delaying the onset of, or causing the regression of a variety of conditions or diseases, including, but not limited to, ocular diseases or conditions. Liquid formulations include, but are not limited to, solutions, suspensions, and in situ gelling formulations.

Described herein are methods, compositions, and liquid formulations for administering rapamycin to a human subject in an amount effective to treat, prevent, inhibit, delay the onset of, or cause the regression of wet AMD.

As described in further detail in the "Detailed Description of the Invention" section, the methods, compositions, and liquid formulations can also be used to deliver a therapeutically effective amount of rapamycin to a subject (including but not limited to a human subject) or to the eyes of a human subject for treating, preventing, suppressing wet AMD, delaying its occurrence, or causing its disappearance. In some variations, the methods, compositions, and liquid formulations are used to treat wet AMD. In some variations, the methods, compositions, and liquid formulations are used to prevent wet AMD. In some variations, the methods and formulations described herein are used to prevent dry AMD from converting to wet AMD. The methods, compositions, and liquid formulations can also be used to deliver a therapeutically effective amount of rapamycin to a subject (including but not limited to a human subject) or to the eyes of a subject for treating, preventing, suppressing CNV, delaying its occurrence, or causing its disappearance. In some variations, the methods, compositions, and liquid formulations can also be used to deliver a therapeutically effective amount of rapamycin to a subject (including but not limited to a human subject) or to the eyes of a subject for treating, preventing, suppressing angiogenesis in the eye, delaying its occurrence, or causing its disappearance. In some variations, the methods, compositions, and liquid formulations are used to treat angiogenesis. Other diseases and conditions for which rapamycin can be used to treat, prevent, inhibit, delay the onset of, or cause regression are described in the "Diseases and Conditions" section of the "Detailed Description of the Invention."

As further described in detail in the "Detailed Description of the Invention" section, the methods, compositions, and liquid formulations can also be used to deliver a therapeutically effective amount of a therapeutic agent other than rapamycin to a subject (including but not limited to a human subject) or to the subject's eye for treating, preventing, inhibiting wet AMD, delaying its occurrence, or causing its regression. In some variations, the methods, compositions, and liquid formulations are used to treat wet AMD. The therapeutic agents that can be used are described in detail in the "Therapeutic Agents" section. Such therapeutic agents include, but are not limited to, compounds that bind to immunophilins. The compounds that can be used to bind to immunophilins include, but are not limited to, the limus family compounds further described in the "Therapeutic Agents" section herein, including rapamycin, SDZ-RAD, tacrolimus, everolimus, pimecrolimus, CCI-779, AP23841, ABT-578, and derivatives, analogs, prodrugs, salts, and esters thereof. The methods, compositions and liquid formulations can also be used to deliver a therapeutically effective amount of a therapeutic agent to a subject (including but not limited to a human subject) or to the subject's eye for treating, preventing, inhibiting CNV, delaying its occurrence, or causing its regression. In some variations, the methods, compositions and liquid formulations are used to treat CNV. The methods, compositions and liquid formulations can also be used to deliver a therapeutically effective amount of a therapeutic agent to a subject (including but not limited to a human subject) or to the subject's eye for treating, preventing, inhibiting angiogenesis in the eye, delaying its occurrence, or causing its regression. In some variations, the methods, compositions and liquid formulations are used to treat angiogenesis. Other diseases and conditions that can be treated, prevented, inhibited, delayed, or caused to resolve using therapeutic agents other than rapamycin are described in the "Diseases and Conditions" section of the "Detailed Description of the Invention."

A kind of liquid preparation described herein comprises a kind of solution, and the solution contains the therapeutic agent dissolved in the solvent.Usually, any solvent with the desired effect can be used, the therapeutic agent is dissolved therein and it can be applied to the subject (including but not limited to human subjects) or applied to the eyes of the subject.Usually, the therapeutic agent of any concentration with the desired effect can be used.In some variations, the preparation is an unsaturated, saturated or supersaturated solution.The solvent can be a pure solvent or can be a mixture of liquid solvent components.In some variations, the solution formed is an in situ gelling preparation.Operable solvents and solution types are well known to those skilled in the art of this type of drug delivery technology.

When placed in rabbit eyes (including but not limited to rabbit eye vitreous body), liquid preparation described herein can form non-divergent agglomerates. In some variations, the non-dispersed agglomerates include gels. In some variations, the liquid preparation comprises therapeutic agent and multiple polymers. In some variations, one of the polymers is polyacrylate or polymethacrylate. In some variations, one of the polymers is polyvinyl pyrrolidone.

In some variations, the non-diverging mass comprises a depot formulation. In some variations, the non-diverging mass consists of a depot formulation.

For liquid formulations that form non-divergent clumps, the non-divergent clumps can generally be of any geometric shape or form. When placed in the vitreous, liquid formulations that form non-divergent clumps can, for example, appear as dense, spherical clumps. In some variations, the liquid formulations described herein, when placed in the vitreous, form milky or white semi-continuous or semisolid non-divergent clumps relative to the medium in which they are placed.

The liquid formulation can generally be administered in any volume that has the desired effect. In one method, a volume of the liquid formulation is administered to the vitreous, and the liquid formulation is less than half the volume of the vitreous.

Routes of administration for administering the liquid formulation that can be used include, but are not limited to, (1) placement (including injection) of the liquid formulation into a medium (including, but not limited to, an aqueous medium in vivo), including, but not limited to, intraocular or periocular injection; or (2) oral administration of the liquid formulation. The liquid formulation can be administered systemically, including, but not limited to, the following routes of administration: rectal, vaginal, instillation, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, intracisternal, epidermal, subcutaneous, intradermal, transdermal, intravenous, intracervical, intraperitoneal, intracranial, intrapulmonary, intrathoracic, intratracheal, nasal, buccal, sublingual, oral, parenteral, or by spray or aerosolization using an aerosol propellant. In some variations, the liquid formulation is administered subconjunctival. In some variations, the liquid formulation is administered intravitreally.

The liquid formulations described herein can be delivered to any medium of a subject (including but not limited to a human subject), including but not limited to an aqueous medium of the subject.

A liquid formulation described herein comprises a liquid formulation of rapamycin or other therapeutic agent. The liquid formulation can include a solution, a suspension, an in situ gelling formulation, or an emulsion. The droplets in the emulsion can generally be of any size, including but not limited to up to about 5,000 nm.

In some formulations described herein, the liquid formulation may comprise a therapeutic agent (including but not limited to rapamycin) and one or more solubilizing agents or solvents. In some variations, the solubilizing agent or solvent is glycerol, DMSO, DMA, N-methylpyrrolidone, ethanol, benzyl alcohol, isopropanol, polyethylene glycols of various molecular weights (including but not limited to PEG300 and PEG400), or propylene glycol, or a mixture of one or more of these substances.

In some formulations described herein, the liquid formulation includes hyaluronic acid.

Liquid preparation described herein can deliver therapeutic agent in the time period of extension.The limiting examples of this type of extended release delivery system is for delivering therapeutic agent to the subject (including but not limited to human subjects) or the system of delivering to the eyes of the subject in sufficient amounts to maintain effective dose in the time period of extension, and the effective dose can treat, prevent, suppress, delay disease or illness in the subject or cause it to disappear.In some variations, liquid preparation is used to treat the disease or illness of the subject (including but not limited to human subjects).In some variations, liquid preparation delivery therapeutic agent continues at least about one month, about two months, about three months, about six months, about nine months or about twelve months.

Liquid preparations described herein can deliver rapamycin or other therapeutic agents over a prolonged time period. A non-limiting example of this type of extended release delivery system is to deliver rapamycin to a subject (including but not limited to human subjects) or to the eyes of a subject in a sufficient amount that can maintain an effective dose over a prolonged time period, and the effective dose can treat, prevent, suppress, delay the generation of wet age-related macular degeneration in the subject or cause it to disappear. In some variations, liquid preparations are used to treat wet age-related macular degeneration over a prolonged time period. In some variations, liquid preparations are used to prevent wet age-related macular degeneration over a prolonged time period. In some variations, liquid preparations are used to prevent dry AMD from being converted into wet AMD over a prolonged time period. In a non-limiting example, liquid preparations deliver rapamycin to the vitreous, sclera, retina, choroid, macula or other tissues of a subject (including but not limited to human subjects) for at least about three months, about six months, about nine months or about twelve months with enough amounts to treat, prevent, suppress, delay the generation of wet age-related macular degeneration or cause it to disappear. In some variations, the level of rapamycin is sufficient to treat AMD. In some variations, the level of rapamycin is sufficient to prevent the development of wet AMD.

Other extended time releases are described in the Detailed Description of the Invention.

Summary of the Figures

1A-1C illustrate the formation of non-dispersed clumps following injection of a liquid formulation into the vitreous of an eye, as is believed to occur in some variations.

Figure 2 depicts rapamycin levels in the vitreous (ng/ml), retina choroid (ng/ml), and sclera (ng/ml) of rabbit eyes 20, 40, 67, and 90 days after subconjunctival injection of 1.256% rapamycin solutions in water, ethanol, and Lutrol® F127.

Figure 3 depicts rapamycin levels in the vitreous (ng/ml), retina choroid (ng/ml), and sclera (ng/ml) of rabbit eyes 14, 35, 62, and 85 days after subconjunctival injection of a 5% rapamycin solution in PEG400 and ethanol.

Figure 4 depicts the levels of rapamycin in the vitreous (ng/ml), retina choroid (ng/ml), and sclera (ng/ml) of rabbit eyes 14, 35, 62, and 90 days after intravitreal injection of a 5% solution of rapamycin in PEG400 and ethanol. Also shown are the levels of rapamycin present in the vitreous (ng/ml) 2 days after injection.

FIG5 depicts images of rabbit eyes 8 days after intravitreal injection of 10 μl ( FIG5A ), 20 μl ( FIG5B ), and 40 μl ( FIG5C ) of a 6% rapamycin suspension in PEG400.

Figure 6 depicts rapamycin levels in the vitreous (ng/ml), retinal choroidal tissue (ng/mg), and sclera (ng/ml) of rabbit eyes 7, 32, 45, and 90 days after subconjunctival injection of 4.2% rapamycin in ethanol, PVPK90, PEG400, and EudragitRL100.

Figure 7 depicts rapamycin levels in the vitreous (ng/ml), retinal choroidal tissue (ng/mg), and sclera (ng/mg) of rabbit eyes 14, 42, 63, and 91 days after subconjunctival injection of a 3% rapamycin suspension in PEG400.

Figure 8 depicts rapamycin levels in the vitreous (ng/ml), retinal choroidal tissue (ng/mg), and sclera (ng/mg) of rabbit eyes 14, 42, 63, and 91 days after intravitreal injection of a 3% rapamycin suspension in PEG400, as well as intravitreal levels 63 and 91 days after injection.

Figure 9 depicts rapamycin levels in the vitreous (ng/ml), retinal choroidal tissue (ng/mg), and sclera (ng/mg) of rabbit eyes 14, 42, 63, and 91 days after subconjunctival injection of 2% rapamycin in ethanol and PEG400.

Figure 10 depicts rapamycin levels in the retinal choroidal tissue (ng/mg) and sclera (ng/mg) of rabbit eyes 14, 42, 63, and 91 days after intravitreal injection of a 2% rapamycin solution in ethanol and PEG400.

Figure 11 depicts rapamycin levels (ng/ml) in the vitreous of rabbit eyes 63 and 91 days after intravitreal injection of a 2% rapamycin solution in ethanol and PEG 400.

Figure 12 depicts the levels of rapamycin in the vitreous (ng/ml) of rabbit eyes 5, 30, 60, 90 and 120 days after subconjunctival injection of 20 μl, 40 μl and 60 μl doses of 2% rapamycin in ethanol and PEG400.

Figure 13 depicts the levels of rapamycin in the retina and choroid (ng/mg) of rabbit eyes 5, 30, 60, 90 and 120 days after subconjunctival injection of 20 μl, 40 μl and 60 μl doses of 2% rapamycin in ethanol and PEG400.

Figure 14 depicts rapamycin levels in the vitreous (ng/ml) of rabbit eyes 5, 30, 60, 90, and 120 days after intravitreal injection of 20 and 40 μl doses of 2% rapamycin in ethanol and PEG400 and 100 μl dose of 0.4% rapamycin in ethanol and PEG400.

Figure 15 depicts rapamycin levels in retinal choroidal tissue (ng/mg) of rabbit eyes 5, 30, 60, 90, and 120 days after intravitreal injection of 20 and 40 μl doses of 2% rapamycin in ethanol and PEG400 and 100 μl dose of 0.4% rapamycin in ethanol and PEG400.

Figure 16 depicts rapamycin levels in the vitreous (ng/ml) of rabbit eyes 5 and 14 days after subconjunctival injection of a single 10 μl dose, a single 60 μl dose, two 30 μl doses, and three 30 μl doses of 2% rapamycin in ethanol and PEG400.

Figure 17 depicts rapamycin levels in the retinal choroidal tissue (ng/mg) of rabbit eyes 5 and 14 days after subconjunctival injection of a single 10 μl dose, a single 60 μl dose, two 30 μl doses, and three 30 μl doses of 2% rapamycin in ethanol and PEG400.

Figure 18 depicts rapamycin levels in the vitreous (ng/ml) of rabbit eyes 5, 14, and 30 days after subconjunctival injection of a single 10 μl dose, a single 30 μl dose, and three 30 μl doses of a 3% rapamycin suspension in PEG400.

Figure 19 depicts rapamycin levels in the retinal choroidal tissue (ng/mg) of rabbit eyes 5, 14, and 30 days after subconjunctival injection of a single 10 μl dose, a single 30 μl dose, and three 30 μl doses of a 3% rapamycin suspension in PEG400.

Figure 20 depicts rapamycin levels in the retinal choroidal tissue (ng/mg) of rabbit eyes 5, 30, and 90 days after intravitreal injection of 10 μl of a 0.2% rapamycin solution in ethanol and PEG400, 10 μl of a 0.6% rapamycin solution in ethanol and PEG400, and 10 μl of a 2% rapamycin solution in ethanol and PEG400.

Figure 21 depicts rapamycin levels in the vitreous (ng/ml) of rabbit eyes 5, 30, and 90 days after intravitreal injection of 10 μl of a 0.2% rapamycin solution in ethanol and PEG400, 10 μl of a 0.6% rapamycin solution in ethanol and PEG400, and 10 μl of a 2% rapamycin solution in ethanol and PEG400.

Figure 22 depicts rapamycin levels in rabbit aqueous humor (ng/ml), cornea (ng/mg), and retinal choroidal tissue (ng/mg) 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days after subconjunctival injection of 40 μl of a 2% rapamycin solution in ethanol and PEG400.

Detailed Description of the Invention

Described herein are compositions, liquid preparations and methods relating to therapeutic agents being delivered to experimenter (including but not limited to human experimenter) or experimenter's eye.These compositions, liquid preparations and methods can be used for treating, preventing, suppressing, delaying eye disease and illness and occur or make it disappear, described eye disease and illness include but not limited to posterior segment disease or illness, include but not limited to choroidal neovascularization, macular degeneration, age-related macular degeneration (including wet AMD and dry AMD), retinal angiogenesis, uveitis and other retinal proliferative disorders.In some modifications, described compositions, liquid preparations and methods are used to treat above-mentioned eye disease or illness.

Described herein are (1) therapeutic agents that can be delivered to a subject (including but not limited to a human subject) or the eye of a subject using the compositions, liquid formulations, and methods described herein; (2) diseases and conditions that can be treated, prevented, inhibited, delayed, or caused to regress by the delivery of therapeutic agents; (3) liquid formulations that can be used to deliver therapeutic agents; (4) routes of administration for delivering liquid formulations; (5) extended delivery of therapeutic agents, including but not limited to rapamycin; and (6) treatments for CNV and wet AMD by delivering rapamycin to a subject (including but not limited to a human subject) or the eye of a subject over an extended period of time using the compositions and liquid formulations described herein.

As used herein, the term "about" refers to the level of accuracy achieved using the methods described herein, such as those in the Examples. However, when an amount of a formulation component is specified, reference to "about" refers to 90-110% of the stated amount.

therapeutic agents

In general, any compounds and compositions known or yet to be discovered that are useful in treating, preventing, inhibiting, delaying the onset of, or causing regression of the diseases and conditions described herein can be therapeutic agents for use in the compositions, liquid formulations, and methods described herein.

The therapeutic agent that can be used includes compounds that work by binding to members of the cellular protein immunophilin family. Such compounds are known as "compounds that bind immunophilin." Compounds that bind immunophilin include, but are not limited to, the "limus" compound family. Examples of usable limus compounds include, but are not limited to, cyclophilin and FK506 binding proteins (FKBPs), including sirolimus (sirolimus) (rapamycin) and its water-soluble analogs SDZ-RAD (Novartis), TAFA-93 (Isotechnika), tacrolimus, everolimus, RAD-001 (Novartis), pimecrolimus, temsirolimus, CCI-779 (Wyeth), AP23841 (Ariad), AP23573 (Ariad) and ABT-578 (Abbott Laboratories). Limus compound analogs and derivatives that can be used include, but are not limited to, compounds described in U.S. Patents 5,527,907; 6,376,517 and 6,329,386 and U.S. Patent Application No. 09/950,307, each of which is incorporated herein by reference in its entirety. Therapeutic agents also include analogs, prodrugs, salts and esters of limus compounds.

The terms rapamycin, rapa, and sirolimus are used interchangeably herein.

Other rapamycin derivatives that can be used include, but are not limited to, 7-epi-rapamycin, 7-thiomethyl-rapamycin, 7-epi-trimethoxyphenyl-rapamycin, 7-epi-thiomethyl-rapamycin, 7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-demethyl-rapamycin, monoester and diester derivatives of rapamycin, rapamycin 27-oxime; 42-oxo analogs of rapamycin; bicyclic rapamycin; rapamycin dimers; rapamycin silyl ethers; rapamycin aryl sulfonates and sulfamates, monoesters and diesters at positions 31 and 42, 30-demethoxyrapamycin, and Vezina et al., "Rapamycin (AY-22,989), A New Antifungal Antibiotic. I. Taxonomy Of The Producing Streptomycete And Isolation Of The Active Principle," J. Antibiot. (Tokyo) 28:721-726 (1975); Sehgal et al., "Rapamycin (AY-22,989), A New AntifungalAntibiotic.II.Fermentation, Isolation And Characterization" J. Antibiot. (Tokyo) 28:727-732 (1975); Sehgal et al., "Demethoxyrapamycin(AY-24,668), A New AntifungalAntibiotic" J. Antibiot. (Tokyo) 36:351-354 (1983); and Paiva et al., "Incorporation OfAcetate, Propionate,And Methionine Into Rapamycin By Streptomyceteshygroscopicus" J Nat Prod54:167-177(1991),WO92/05179,EP467606, Caufield et al., “Hydrogenated Rapamycin Derivatives,” U.S. Pat. No. 5,023,262; Kao et al., “Bicyclic Rapamycins,” U.S. Pat. No. 5,120,725; Kao et al., “Rapamycin Dimers,” U.S. Pat. No. 5,120,727; Failli et al., “Silyl Ethers of Rapamycin,” U.S. Pat. No. 5,120,842; Failli et al., “Rapamycin 42-Sulfonates And 42-(N-carboalkoxy) Sulfamates Useful As Immunosuppressive Agents,” U.S. Pat. No. 5,177,203; Nicolaou et al., “Total Synthesis Of Rapamycin,” J. Am. Chem. Soc. 115:4419-4420 (1993); Romo et al., “Total Synthesis Of (-)Rapamycin Using An Evans-Tishchenko Fragment Coupling" J. Am. Chem. Soc. 115: 7906-7907 (1993); and other derivatives described in Hayward et al., "Total Synthesis Of Rapamycin Via A Novel Titanium-Mediated Aldol Macrocyclization Reaction" J. Am. Chem. Soc., 115: 9345-9346 (1993), each of which is incorporated herein by reference in its entirety.

Limus family compounds can be used to treat, prevent, inhibit, delay angiogenesis-mediated eye diseases and conditions (including choroidal neovascularization) or to eliminate compositions, liquid formulations and methods. The limus compound family can be used to prevent, treat, inhibit, delay AMD (including wet AMD) or to eliminate it. Rapamycin and rapamycin derivatives and analogs can be used to prevent, treat, inhibit, delay angiogenesis-mediated eye diseases and conditions (including choroidal neovascularization) or to eliminate it. Rapamycin can be used to prevent, treat, inhibit, delay AMD (including wet AMD) or to eliminate it. In some variations, members of the limus compound family or rapamycin are used to treat wet AMD or angiogenesis-mediated eye diseases and conditions, including choroidal neovascularization.

Other therapeutic agents that may be used include those disclosed in the following patents and publications, each of which is incorporated herein by reference in its entirety: PCT Publication WO 2004/027027, published April 1, 2004, entitled Method of Inhibiting Choroidal Neovascularization, assigned to Trustees of the University of Pennsylvania; U.S. Patent No. 5,387,589, issued February 7, 1995, entitled Method of Treating Ocular Inflammation, inventor Prassad Kulkarni, assigned to the University of Louisville Research Foundation; U.S. Patent No. 6,376,517, issued April 23, 2003, entitled Pipecolic Acid Derivatives for Vision and Memory Disorders, assigned to GPI NIL Holdings, Inc.; PCT Publication WO 2004/028477, published April 8, 2004, entitled Method of Subretinal Administration of Therapeutics Including Steroids: method for localizing pharmadynamic action at the choroid and retina; and related methods for treatment and or prevention of retinal diseases, assigned to Innorx, Inc.; U.S. Patent No. 6,416,777, filed July 9, 2002, entitled Ophthalmic drug delivery device, assigned to Alcon Universal Ltd; U.S. Patent No. 6,713,081, issued March 30, 2004, entitled Ocular therapeutic agent delivery device and methods for making and using such devices, assigned to the Department of Health and Human Services; U.S. Patent No. 5,100,899, issued March 31, 1992, entitled Methods of inhibiting transplant rejection in mammals using rapamycin and derivatives and prodrugs thereof.

Other therapeutic agents that may be used include pyrrolidines, dithiocarbamates (NFκB inhibitors); squalamine; TPN470 analogs and fumagillin; PKC (protein kinase C) inhibitors; Tie-1 and Tie-2 kinase inhibitors; VEGF receptor kinase inhibitors; proteosome inhibitors such as Velcade ^™ (bortezomib) for injection; ranibuzumab (Lucentis ^™ ) and other antibodies to the same target; pegaptanib (Macugen ^™) ); vitronectin receptor antagonists, such as cyclic peptide antagonists of vitronectin receptor-type integrins; α-v/β-3 integrin antagonists; α-v/β-1 integrin antagonists; thiazolidinediones, such as rosiglitazone or troglitazone; interferons, including gamma-interferon or interferons targeted to CNV through the use of dextran and metal coordination; pigment epithelium-derived factor (PEDF); endostatin; angiostatin; tumistatin; canstatin; anecortave acetate; acetonide; triamcinolone; tetrathiomolybdate; RNA silencing or RNA interference (RNAi) of angiogenic factors, including ribozymes targeting VEGF expression; Accutane ^™ (13-cis retinoic acid); ACE inhibitors, including but not limited to quinopril, captopril, and perindozril; mTOR inhibitors (mammalian target of rapamycin); 3-aminothalidomide; pentoxifylline; 2-methoxyestradiol; colchicine; AMG-1470; cyclooxygenase inhibitors, such as nepafenac, rofecoxib, diclofenac, rofecoxib, NS398, celecoxib, vioxx, and (E)-2-alkyl-2(4-methylsulfonylphenyl)-1-phenylethylene; t-RNA synthase modulators; metalloproteinase 13 inhibitors; acetylcholinesterase inhibitors; potassium channel blockers; endorepellin; purine analogs of 6-thioguanine; cycloperoxide ANO-2; (recombinant) arginine deiminase; epigallocatechin-3-gallate; cerivastatin; suramin analogs; VEGF trap molecules; apoptosis inhibitors; Visudyne ^™ , snET2, and other photosensitizers, which can be used in photodynamic therapy (PDT); hepatocyte growth factor inhibitors (growth factor antibodies or their receptors, small molecule inhibitors of c-met tyrosine kinase, truncated forms of HGF such as HK4).

Other therapeutic agents that can be used include anti-inflammatory agents, including but not limited to nonsteroidal anti-inflammatory agents and steroidal anti-inflammatory agents. In some variations, the active agent that can be used in the liquid preparation is ace-inhibitor, endogenous cytokine, the active agent that affects basement membrane, the active agent that affects endothelial cell growth, adrenergic agonist or blocker, cholinergic agonist or blocker, aldose reductase inhibitor, analgesic, anesthetic, antiallergic agent, antibacterial drug, antihypertensive drug, pressor, antiprotozoal agent, antiviral agent, antifungal agent, anti-infective agent, antitumor agent, antimetabolite and antiangiogenic agent.

Steroidal therapeutic agents that may be used include, but are not limited to, 21-pregnenolone acetate, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednol, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl ester butyl), fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednoletabonate, maprednione mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone caproate hexacetonide) and any derivatives thereof.

In some variations, cortisone, dexamethasone, fluocinolone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone and their derivatives may be used. The liquid formulation may comprise a combination of two or more steroid therapeutic agents.

In one non-limiting example, the steroid therapeutic agent can comprise from about 0.05% to about 50% by weight of the liquid formulation. In another non-limiting example, the steroid comprises from about 0.05% to about 10%, from about 10% to 20%, from about 30% to about 40%, or from about 40% to about 50% by weight of the liquid formulation.

Other non-limiting examples of therapeutic agents that can be used include, but are not limited to, anesthetics, analgesics, cell transport/migration inhibitors such as colchicine, vincristine, cytochalasin B, and related compounds; carbonic anhydrase inhibitors such as acetazolamide, methazolamide, dichlorphenamide, diamox, and neuroprotectants such as nimodipine and related compounds; antibiotics such as tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol, rifampicin, ciprofloxacin, aminoglycosides, gentamicin, erythromycin, and penicillins, quinolones, ceftazidime, vancomycin, imine, and penicillins; imipeneme; antifungals such as amphotericin B, croconazole, ketoconazole, and miconazole; antibacterials such as sulfonamides, sulfadiazine, sulfacetamide, sulfamethizole, and sulfisoxazole, nitrofurazone, and sodium propionate; antivirals such as idoxuridine, trifluorothymidine, trifluorouridine, acyclovir, ganciclovir, cidofovir, interferon, DDI, AZT, foscamet, vidarabine, irbavirin, protease inhibitors, and anticytomegalovirus agents; antiallergics such as sodium cromolyn. cromoglycate), antazoline, methapyriline, chlorpheniramine, cetirizine, pyrilamine, and prophenpyridamine; synthetic glucocorticoids and mineralocorticoids and more common hormones derived from cholesterol (DHEA, progesterone, estrogens); nonsteroidal anti-inflammatory drugs, such as salicylates, indomethacin, ibuprofen, diclofenac, flurbiprofen, piroxicam, and COX2 inhibitors; antineoplastic agents such as carmustine, cisplatin, fluorouracil; doxorubicin, asparaginase, azacitidine, azathioprine, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cyclophosphamide, cyclosporine, cytarabine, dacarbazine, dactinomycin, daunorubicin daunorubicin, doxorubicin, estramustine, etoposide, etretinate, filgrastin, floxuridine, fludarabine, fluorouracil, florxymesterone, flutamide, goserelin, hydroxyurea, ifosfamide, leuprolide, levamisole, limustine, nitrogen mustard, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, plicamycin plicamycin, procarbazine, sargramostin, streptozocin, tamoxifen, taxol, teniposide, thioguanine, uracil mustard, vinblastine, vincristine, and vindesine; immunotherapy drugs such as vaccines and immunostimulants; insulin, calcitonin, parathyroid hormone, and peptides and vasopressin-releasing factor; beta-adrenergic blocking agents such as timolol, levobunolol, and betaxolol betaxolol; cytokines, interleukins and growth factors, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor beta, ciliary neurotrophic growth factor, glial cell line-derived neurotrophic factor, NGF, EPO, PLGF, brain nerve growth factor (BNGF), vascular endothelial growth factor (VEGF), and monoclonal antibodies or fragments thereof to such growth factors; anti-inflammatory agents such as hydrocortisone, dexamethasone, fluocinolone, prednisone, prednisolone, methylprednisolone, fluorometholone, betamethasone, and triamcinolone; decongestants such as phenylephrine, naphazoline, and tetrahydrazoline; miotics and anticholinesterases such as pilocarpine, carbachol, diisopropyl fluorophosphate, and acetaminophen. fluorophosphates), phospholineiodine, and demecarium bromide; mydriatics such as atropine sulfate, cyclopentolate, homatropine, scopolamine, tropicamide, and eucatropine; sympathomimetics such as epinephrine and vasoconstrictors and vasodilators; anticoagulants such as heparin, antifibrinogen, fibrinolysin, and anticlotting agents. activase), antidiabetic agents including acetohexamide, chlorpropamide, glipizide, glyburide, tolazamide, tolbutamide, insulin and aldose reductase inhibitors, hormones, peptides, nucleic acids, sugars, lipids, glycolipids, glycoproteins and other macromolecules, including endocrine hormones such as vasopressin, insulin, insulin-related growth factor, thyroxine, growth hormone; heat shock proteins; immune response modifiers such as muramyl dipeptide, cyclosporine, interferons (including α-, β- and γ-interferons), interleukin 2, cytokines, FK506 (an epoxy-pyrido-oxazaheterocyclic tricosene-tetraone, also known as tacrolimus), tumor necrosis factor, pentostatin, thymopentin, transforming factor β2, erythropoetin; anti-neoplastic proteins (e.g., anti-VEGF, interferon), antibodies (monoclonal, polyclonal, humanized, etc.) or antibody fragments, oligomeric aptamers, aptamers and gene fragments (oligonucleotides, plasmids, ribozymes, small interfering RNA (siRNA), nucleic acid fragments, peptides), immunomodulators such as endoxan, thalidomide, tamoxifen; antithrombotic and vasodilator drugs such as rtPA, urokinase, plasmin; nitric oxide donors, nucleic acids, dexamethasone, cyclosporine A, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., folinic acid, edatrexate, methotrexate, pirtrexim, pteropterin, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiabendazole), In some variations, the immunosuppressant is dexamethasone. In some variations, the immunosuppressant is cyclosporine A.

In some variations, the formulation comprises a combination of one or more therapeutic agents.

Other non-limiting examples of therapeutic agents that can be used in the formulations described herein include antibacterial antibiotics, aminoglycosides (e.g., amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, tobramycin, trospectomycin), amphenicols (e.g., azidamfenicol, chloramphenicol, florfenicol, thiamphenicol), ansamycins (e.g., rifamide, rifampicin, rifamycin SV, rifapentine, rifaximin), beta-lactams (e.g., carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem, imipenem, meropenem, panipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefmandole, cefatrizine, cefazolin), cefazedone, cefazolin, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime cefepime, cefetamet, cefixime, cefinenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefpodoximeproxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime (ceftizoxime), ceftriaxone, cefuroxime, cefuzonam, cephacetrilesodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin, cephalothin, cephapirin sodium sodium), cephradine, pivcefalexin), cephamycins (e.g., cefbuperazone, cefinetazole, cefminox, cefotetan, cefoxitin), monobactams (e.g., aztreonam, carumonam, tigemonam), oxacephems (flomoxef, moxalactam), penicillins (e.g., amdinocillin, amdinocillinpivoxil), amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin azlocillin, bacampicillin, benzylpenicillin acid, benzylpenicillin sodium, carbenicillin, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, fluxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamcillin, penethamate hydriodide, Penicillin G Benethamine, Penicillin G Benzathine, Penicillin G Dibenzamide, Penicillin G Calcium, Penicillin G Hydrate, Penicillin G Potassium, Penicillin G Procaine, Penicillin N, Penicillin O, Penicillin V, Penicillin V Benzathine, Penicillin V Hydrate, Penimepicycline, Phenethicillin Potassium potassium), piperacillin, pivampicillin, propicillin, quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin, ticarcillin), ritipenem, lincosamides (e.g., clindamycin, lincomycin), macrolides (e.g., azithromycin, carbomycin, clarithromycin, dirithromycin, erythromycin, erythromycin acistrate, erythromycin estolate, erythromycin gluceptate, glucoheptonate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, josamycin, leucomycins, midecamycin, miokamycin, oleandomycin, primycin, rokitamycin, rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides (e.g., amphomycins, bacitracin, capreomycin, colistin, enduracidin, enviromycin), enviomycin, fusafungine, gramicidins, gramicidins, micamycin, polymyxins, pristinamycin, ristocetin, teicoplanin, thiostrepton, tuberactinomycin, tyrocin, gramicidin, vancomycin, puromycin, virginiamycin, zinc bacitracin), tetracyclines (e.g., apicycline apicycline, chlortetracycline, clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, pyricycline, pipacycline, rolitetracycline, sancycline, tetracycline, and others (e.g., cycloserine, mupirocin, tuberin); synthetic antibacterial drugs, 2,4-diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone) furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin, nadifloxacin, nalidixic acid, acid), norfloxacin, ofloxacin, oxolinic acid, pazufloxacin, pefloxacin, pipemidic acid, piromidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, chloramine-B, chloramine-T, dichloramine-T, N2-formylsulfisomidine, N4-β-d-glucosylsulfonamide, mafenide, 4′-(methylsulfamoyl)sulfonamide), sulfanilanilide, noprylsulfamide, phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfadiazole sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene, sulfaloxicacid, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sulfametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, 4-sulfanilamidosalicylic acid, n4-sulfanilylsulfanilamide, sulfanilylurea, n-Sulfanilide-3,4-propylglutamine, sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide, sulfisomidine, sulfisoxazole), sulfones (e.g., acedapsone, dapsone acetic acid) acediasulfone, acetosulfone sodium, dapsone, diathymosulfone, glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid, sulfanilamide, sulfoxone sodium, thiazolsulfone) and others (e.g., clofoctol, hexedine, methenamine, methenamine anhydromethylene-citrate, methenamine hippurate, methenamine mandelate, methenamine sulfosalicylate, nitroxoline, taurolidine taurolidine, xibomol), antifungal antibiotics, polyenes (e.g., amphotericin B, candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin), azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin, xicanin siccanin, tubercidin, viridin, synthetic antifungals, allylamines (e.g., butenafine, naftifine, terbinafine), imidazoles (e.g., bifonazole, butoconazole, chlordantoin, chlormidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazole nitrate, sertaconazole sertaconazole, sulconazole, tioconazole), thiocarbamates (e.g., tolciclate, tolindate, tolnaftate), triazoles (e.g., fluconazole, itraconazole, saperconazole, terconazole), acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, ciclopirox, clooxyquin, coparaffinate, diamthazole dihydrochloride, exalarnide, flucytosine, chlorphenesin, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine, chlorpheniramine halethazole, hexetidine, loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione, salicylanilide, sodium propionate, sulbentine, tenonitrozole, triacetin, ujothion, undecylenic acid, zinc propionate, antineoplastic agents, antibiotics and analogs (e.g., aclacinomycins, actinomycin F1, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycin mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), antimetabolites (e.g., folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6- mercaptopurine, thiamiprine, thioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine fluoxuridine, fluorouracil, gemcitabine, tagafur), anti-inflammatory agents, steroidal anti-inflammatory agents, acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, cloprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazole, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, glycyrrhetinic acid, fluazacort, flucloxacil, flumethasone, flunisolide, fluocinolone, fluocinolone acetate, fluocinolone butyl, fluocortolone, fluorometholone, methylfluanilide acetate, fluoromethylidene acetate, fluprednisolone, flu Hydrocortisone, fluticasone propionate, flumethasone, halcinonide, halobetasol propionate, halometasone, halopredazone acetate, hydrocortisone, hydrocortisone, loteprednol ethyl carbonate, maprednione, medrysone, methylprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednisolone valerate, prednidine, rimexolone, tixoxolone tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone hexacetonide, nonsteroidal anti-inflammatory agents, aminoaryl carboxylic acid derivatives (e.g., phenylethylanthranilic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, acid), mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), aryl acetate derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetinguacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, triflate isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (e.g., bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpropionic acid derivatives (e.g., alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen), indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (e.g., difenamizole, epirizole), pyrazolones (e.g., apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, isopropyl antipyrine propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (e.g., acetaminosalol, aspirin, benoylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, acetylsalicylate), phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine sulfasalazine), thiazine carboxamides (e.g., ampiroxicam, droxicam, isoxicam, lomoxicam, piroxicam, tenoxicam), ε-ethylaminocaproic acid, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, a-bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, nimesulide, oxaceprol (oxaceprol), paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, and zileuton.

The therapeutic agent may also be used in combination with other therapeutic agents and therapies, including but not limited to therapeutic agents and therapies for treating, preventing, inhibiting, delaying, or regressing angiogenesis or neovascularization (particularly CNV). In some variations, an additional therapeutic agent or therapy is used to treat or regress angiogenesis or neovascularization (particularly CNV). Non-limiting examples of such additional therapeutic agents and therapies include pyrrolidine, dithiocarbamate (NFκB inhibitor); squalamine; TPN470 analogs and fumagillin; PKC (protein kinase C) inhibitors; Tie-1 and Tie-2 kinase inhibitors; VEGF receptor kinase inhibitors; proteosome inhibitors such as Velcade ^™ (bortezomib for injection; ranibuzumab (Lucentis ^™ ) and other antibodies to the same target; pegaptanib (Macugen ^™) ); vitronectin receptor antagonists, such as cyclic peptide antagonists of vitronectin receptor-type integrins; α-v/β-3 integrin antagonists; α-v/β-l integrin antagonists; thiazolidinediones, such as rosiglitazone or troglitazone; interferons, including gamma-interferon or interferons targeted to CNV through the use of dextran and metal coordination; pigment epithelium-derived factor (PEDF); endostatin; angiostatin; tumistatin; canstatin; anecortave acetate; acetonide; triamcinolone; tetrathiomolybdate; RNA silencing or RNA interference (RNAi) of angiogenic factors, including ribozymes targeting VEGF expression; Accutane ^™ (13-cis retinoic acid); ACE inhibitors, including but not limited to quinopril, captopril, and perindozril; mTOR inhibitors (mammalian target of rapamycin); 3-aminothalidomide; pentoxifylline; 2-methoxyestradiol; colchicine; AMG-1470; cyclooxygenase inhibitors, such as nepafenac, rofecoxib, diclofenac, rofecoxib, NS398, celecoxib, vioxx, and (E)-2-alkyl-2(4-methylsulfonylphenyl)-1-phenylethylene; t-RNA synthase modulators; metalloproteinase 13 inhibitors; acetylcholinesterase inhibitors; potassium channel blockers; endorepellin; purine analogs of 6-thioguanine; cycloperoxide ANO-2; (recombinant) arginine deiminase; epigallocatechin-3-gallate; cerivastatin; suramin analogs; VEGF trap molecules; hepatocyte growth factor inhibitors (growth factor antibodies or their receptors, small molecule inhibitors of c-met tyrosine kinase, truncated forms of HGF such as HK4); apoptosis inhibitors; Visudyne ^™ , snET2, and other photosensitizers for photodynamic therapy (PDT); and laser photocoagulation.

Diseases and conditions that can be treated, prevented, inhibited, delayed, or caused to relapse

The present invention describes diseases and conditions that can be treated, prevented, inhibited, delayed in onset, or caused to resolve using the therapeutic agents and formulations, liquid formulations, and methods described herein. In some variations, the diseases and conditions are treated using the therapeutic agents and formulations, liquid formulations, and methods described herein. Unless otherwise specified herein, all treatment methods are considered to be performed on subjects including, but not limited to, human subjects.

In general, any eye disease or condition susceptible to treatment, prevention, inhibition, delayed onset, or caused regression using the therapeutic agents and formulations, liquid formulations, and methods described herein can be treated, prevented, inhibited, delayed onset, or caused regression. Examples of eye diseases or conditions include, but are not limited to, diseases or conditions associated with neovascularization, including retinal and/or choroidal neovascularization.

Diseases or conditions associated with retinal and/or choroidal neovascularization that can be treated, prevented, inhibited, delayed, or caused to regress using the formulations, liquid formulations, and methods described herein include, but are not limited to, diabetic retinopathy, macular degeneration, wet and dry AMD, retinopathy of prematurity (retrolental fibroplasia), infections causing retinitis or choroiditis, presumed ocular histoplasmosis, myopic degeneration, angioid streaks, and ocular trauma. Other non-limiting examples of ocular diseases and conditions that can be treated, prevented, inhibited, delayed, or caused to regress using the formulations, liquid formulations, and methods described herein include, but are not limited to, pseudoxanthoma elasticum, vein occlusion, artery occlusion, carotid obstructive disease, sickle cell anemia, Eales disease, myopia, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma, polypoidal choroidal vasculopathy, post-laser complications, complications of idiopathic central serous chorioretinopathy, complications of choroidal inflammatory conditions, erythema, The following diseases or conditions are considered to be cataracts: rubeosis, diseases associated with redness (neovascular formation in the canthus), neovascular glaucoma, uveitis and chronic uveitis, macular edema, proliferative retinopathy, and diseases or conditions caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy (including postoperative proliferative vitreoretinopathy), whether or not associated with diabetes mellitus.

In some variations, preparations described herein and pharmaceutical preparations are used to prevent or delay the generation of ocular diseases or illness, wherein subject (including but not limited to human subject) is under the risk of the raising of ocular diseases or illness. The subject with the risk of disease or illness raising is a subject with one or more signs, and this disease or illness is likely to occur in this particular subject. In some variations, there is a subject with the risk of raising wet AMD occurring for at least one eye to suffer from dry AMD. In some variations, there is a subject with the risk of raising wet AMD occurring for another eye to suffer from wet AMD. In some variations, preparations described herein and pharmaceutical preparations are used to prevent or delay CNV generation in the subject under the risk of raising CNV occurring, including but not limited to preventing or delaying the generation of CNV in subject (including but not limited to human subject whose one eye suffers from AMD) fellow eye. In some variations, preparations described herein and pharmaceutical preparations are used to prevent or delay the generation of CNV in the fellow eye of one eye to suffer from wet AMD patient. In some variations, preparations and pharmaceutical preparations comprise limus compounds, including but not limited to rapamycin. In some variations, the formulations and pharmaceutical formulations are administered periocularly (including but not limited to, subconjunctivally) to a human subject having 20/40 or better vision. In some variations, the formulations and pharmaceutical formulations are administered periocularly (including but not limited to, subconjunctivally) to an eye of a human subject, wherein the eye to which the formulation is administered has 20/40 or better vision.

In some variations, preparations described herein and pharmaceutical preparations are used to treat, prevent or delay AMD. In some variations, preparations described herein and pharmaceutical preparations are used to treat, prevent or delay dry AMD. In some variations, preparations described herein or pharmaceutical preparations are used to treat, prevent or delay the generation of central geographic atrophy to the experimenter (including but not limited to human experimenter) suffering from non-central geographic atrophy. In some variations, preparations and pharmaceutical preparations contain limus compounds, including but not limited to rapamycin. In some variations, preparations and pharmaceutical preparations periocular (including but not limited to subconjunctival) are applied to the human experimenter with 20/40 or better vision. In some variations, preparations described herein and pharmaceutical preparations are applied, and experimenter (including but not limited to human experimenter) is also used with another therapy treatment for the treatment of disease or illness. In some variations, the formulations and pharmaceutical formulations described herein are used to treat, prevent, or delay the onset of wet or dry AMD, and the subject (including but not limited to a human subject) is also treated with laser therapy (such as photodynamic laser therapy) before, during, or after treatment with the formulations and pharmaceutical formulations described herein.

In some variations, the formulations and pharmaceutical formulations described herein are used to treat one or more of uveitis, allergic conjunctivitis, macular edema, glaucoma, or dry eye.

In some variations, a formulation or pharmaceutical formulation comprises a limus compound such as rapamycin and is administered to treat, prevent, or delay the onset of dry eye. In some variations, a formulation or pharmaceutical formulation comprises a limus compound such as rapamycin and is administered to treat, prevent, or delay the onset of allergic conjunctivitis.

In some variations, the formulations and pharmaceutical preparations described herein are used to treat glaucoma. In some variations, the formulations and pharmaceutical preparations described herein for treating glaucoma contain limus compounds such as rapamycin and are used as surgical adjuvants to prevent, alleviate or delay surgical complications. In some variations, the formulations and pharmaceutical preparations described herein for treating glaucoma contain limus compounds such as rapamycin and are used to improve or prolong the success of surgical implants. In some variations, the formulations and pharmaceutical preparations described herein for treating glaucoma contain limus compounds such as rapamycin and are used to promote or prolong the success of argon laser trabeculectomy or other glaucoma-related surgeries. In some variations, the formulations and pharmaceutical preparations described herein have neuroprotective effects and are used to treat glaucoma.

In some variations, the formulations and pharmaceutical preparations described herein are used to treat retinitis pigmentosa. In some variations, the formulations and pharmaceutical preparations described herein for treating glaucoma comprise limus compounds such as rapamycin and are used to treat, prevent, or delay the onset of retinitis pigmentosa. In some variations, the formulations and pharmaceutical preparations described herein have neuroprotective effects and are used to treat retinitis pigmentosa.

In some variations, the formulations and pharmaceutical preparations described herein are used to treat one or more central retinal vein occlusive disease (CRVO), branch retinal vein occlusion (BRVO), retinal vascular diseases and conditions, macular edema, diabetic macular edema, scleral neovascularization, diabetic retinopathy, or corneal transplant rejection. In some variations, the formulations and pharmaceutical preparations contain a limus compound such as rapamycin and are administered to treat, prevent, or delay the onset of one or more of these diseases or conditions. In some variations, the formulations and pharmaceutical preparations described herein are administered subconjunctival to an eye with 20/40 or better vision.

When used to treat, prevent, inhibit, delay the onset of, or resolve uveitis, the formulations and liquid formulations described herein can be administered by a variety of routes known in the art, including, but not limited to, ocular or oral administration. Other routes of administration are known and conventional in the art. In some variations, the formulations described herein comprise rapamycin and are used to treat uveitis.

A kind of disease that uses preparation described herein, liquid preparation and method to treat, suppress, delay its generation or its disappearance is wet AMD.In some modifications, use preparation described herein, liquid preparation and method to treat wet AMD.Wet AMD is characterized by blood vessel growing into undesirable position under retina from their common position in choroid.These new blood vessels leak and hemorrhage and cause visual acuity to reduce, and may go blind.

The formulations, liquid formulations, and methods described herein can also be used to prevent or delay the conversion of dry AMD (in which the retinal pigment epithelium, or RPE, degenerates and causes photoreceptor cell death and the formation of yellow deposits called drusen under the retina) to wet AMD.

"Macular degeneration" is characterized by an overproduction of fibrillar deposits in the macula and retina and atrophy of the retinal pigment epithelium. As used herein, an eye "afflicted" by macular degeneration is understood to mean that the eye displays at least one detectable physiological characteristic associated with a macular degenerative disease. Administration of rapamycin has been shown to limit and regress angiogenesis, such as choroidal neovascularization in age-related macular degeneration (AMD), which occurs without treatment. The term "angiogenesis," as used herein, refers to the production of new blood vessels in a tissue or organ ("neovascularization"). An "angiogenesis-mediated disease or condition" of the eye or retina is a disease or condition in which new blood vessels form in the eye or retina in a pathogenic manner, causing decreased or loss of vision or other problems, such as choroidal neovascularization associated with AMD.

The formulations and liquid formulations described herein (including but not limited to formulations and liquid formulations containing rapamycin) can also be used to treat, prevent, inhibit, delay the occurrence of, or cause the regression of a variety of immune-related diseases and conditions, including but not limited to organ transplant rejection, graft-versus-host disease, autoimmune diseases, inflammatory diseases, hyperproliferative vascular diseases, solid tumors, and fungal infections in a host. In some variations, the formulations and liquid formulations described herein (including but not limited to formulations and liquid formulations containing rapamycin) are used to treat a variety of immune-related diseases and conditions, including but not limited to organ transplant rejection, graft-versus-host disease, autoimmune diseases, inflammatory diseases, hyperproliferative vascular diseases, solid tumors, and fungal infections in a host. The formulations and liquid formulations described herein (including but not limited to formulations and liquid formulations containing rapamycin) can be used as immunosuppressants. The formulations and liquid formulations described herein (including but not limited to formulations and liquid formulations containing rapamycin) can be used to treat, prevent, inhibit, or delay the occurrence or regression of rejection of a transplanted organ or tissue, including but not limited to a transplanted heart, liver, kidney, spleen, lung, small intestine, pancreas, and bone marrow. In some variations, the formulations and liquid formulations described herein are used to treat the occurrence of rejection of a transplanted organ or tissue, including but not limited to a transplanted heart, liver, kidney, spleen, lung, small intestine, pancreas, and bone marrow. When used to treat, prevent, inhibit, delay the occurrence or regression of an immune-related disease, including but not limited to transplant rejection, the formulations and liquid formulations described herein can be administered by a variety of routes known in the art, including but not limited to oral administration.

Systemic administration can be accomplished by oral liquid formulations. Other routes of systemic administration are conventional in the art. Some examples are listed in the detailed description of the invention.

As used herein, "inhibiting" a disease or condition by administering a therapeutic agent means that the development of at least one detectable physiological characteristic or symptom of the disease or condition is slowed or halted following administration of the therapeutic agent, compared to the progression of the disease or condition in the absence of administration of the therapeutic agent.

As used herein, "preventing" a disease or condition by administering a therapeutic agent is the absence of development of detectable physiological characteristics or symptoms indicative of the disease or condition following administration of the therapeutic agent.

As used herein, "delaying the onset" of a disease or condition by administering a therapeutic agent means that after administration of the therapeutic agent, at least one detectable physiological characteristic or symptom of the disease or condition develops later than the progression of the disease or condition in the absence of administration of the therapeutic agent.

As used herein, "treating" a disease or condition by administering a therapeutic agent means that the progression of at least one detectable physiological characteristic or symptom of the disease or condition is slowed, halted, or reversed following administration of the therapeutic agent, compared to the progression of the disease or condition without administration of the therapeutic agent.

As used herein, "causing regression" of a disease or condition by administration of a therapeutic agent means that the progression of at least one detectable physiological characteristic or symptom of the disease or condition is reversed to some extent following administration of the therapeutic agent.

Subjects (including but not limited to human subjects) with a predisposition or need for prevention can be determined by a skilled physician using methods and criteria established in the art according to the teachings herein. A skilled physician can also readily diagnose individuals in need of inhibition or treatment based on established criteria for identifying angiogenesis and/or neovascularization according to the teachings herein.

As used herein, a "subject" is generally any animal that may benefit from administration of a therapeutic agent described herein. In some variations, the therapeutic agent is administered to a mammalian subject. In some variations, the therapeutic agent is administered to a human subject. In some variations, the therapeutic agent may be administered to a veterinary animal subject. In some variations, the therapeutic agent may be administered to a model laboratory animal subject.

Other diseases and conditions that can be treated, prevented, inhibited, delayed in onset, or caused to regress using the methods described herein include those disclosed in the following patents and publications, the contents of each of which are incorporated herein by reference in their entirety: PCT Publication WO 2004/027027, published April 1, 2004, entitled Method of inhibiting choroidal neovascularization, assigned to the Trustees of the University of Pennsylvania; U.S. Patent No. 5,387,589, issued February 7, 1995, entitled Method of Treating Ocular Inflammation, inventor Prassad Kulkarni, assigned to the University of Louisville Research Foundation; U.S. Patent No. 6,376,517, issued April 23, 2003, entitled Pipecolic acid derivatives for vision and memory disorders, assigned to GPI NIL Holdings, Inc; PCT Publication WO 2004/028477, published April 8, 2004, entitled Method of subretinal administration of therapeutics including steroids: method for localizing pharmadynamic action at the choroid and retina; and related methods for treatment and or prevention of retinal diseases, assigned to Innorx, Inc.; U.S. Patent No. 6,416,777, issued July 9, 2002, entitled Ophthalmic drug delivery device, assigned to Alcon Universal Ltd; U.S. Patent No. 6,713,081, issued March 30, 2004, entitled Ocular therapeutic agent delivery device and methods for making and using such devices, assigned to the Department of Health and Human Services; and U.S. Patent No. 5,536,729, filed July 16, 1996, entitled Rapamycin Formulations for Oral Administration, assigned to American Home Products Corp., and U.S. Patent Application Nos. 60/503,840 and 10/945,682.

Liquid preparations

The liquid formulations described herein contain a therapeutic agent and can generally be any liquid formulation, including but not limited to solutions, suspensions, and emulsions. In some variations, the liquid formulation forms a non-dispersed mass relative to the surrounding medium when placed in the vitreous of a rabbit eye.

When administering a volume of liquid formulation, it should be understood that the accuracy of the various devices used to administer liquid formulations has a certain degree of imprecision. When a volume is specified, it should be understood that this is the intended volume. However, some devices, such as insulin syringes, have inaccuracies greater than 10%, and sometimes inaccuracies greater than 20% or more. Hamilton HPLC-type syringes are generally considered accurate within 10% and are recommended for injection volumes less than 10 μl.

In some variations, the volume of the liquid formulations described herein administered to the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is less than about 500 μl, less than about 400 μl, less than about 300 μl, less than about 200 μl, less than about 100 μl, less than about 90 μl, less than about 80 μl, less than about 70 μl, less than about 60 μl, less than about 50 μl, less than about 40 μl, less than about 30 μl, less than about 20 μl, less than about 10 μl, less than about 5 μl, less than about 3 μl, or less than about 1 μl. In some variations, the volume of the liquid formulations described herein administered to the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is less than about 20 μl. In some variations, the volume of the liquid formulations described herein administered to the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is less than about 10 μl. In some variations, the volume of the liquid formulation described herein administered to the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 0.1 μl and about 200 μl, between about 50 μl and about 200 μl, between about 50 μl and about 150 μl, between about 0.1 μl and about 100 μl, between about 0.1 μl and about 50 μl, between about 1 μl and about 40 μl, between about 1 μl and about 30 μl, between about 1 μl and about 20 μl, between about 1 μl and about 10 μl, or between about 1 μl and about 5 μl. In some variations, the volume of the liquid formulation described herein administered to the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 10 μl. In some variations, the volume of a liquid formulation described herein administered into the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 5 μl. In some variations, the volume of a liquid formulation described herein administered into the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 5 μl. In some variations, the volume of a liquid formulation described herein administered into the vitreous of a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 0.1 μl and about 200 μl.

In some variations, the total volume of a liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is less than about 1000 μl, less than about 900 μl, less than about 800 μl, less than about 700 μl, less than about 600 μl, less than about 500 μl, less than about 400 μl, less than about 300 μl, less than about 200 μl, less than about 100 μl, less than about 90 μl, less than about 80 μl, less than about 70 μl, less than about 60 μl, less than about 50 μl, less than about 40 μl, less than about 30 μl, less than about 20 μl, less than about 10 μl, less than about 5 μl, less than about 3 μl, or less than about 1 μl. In some variations, the volume of a liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is less than about 20 μl. In some variations, the volume of a liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye, including but not limited to a human subject's eye, is less than about 10 μl. In some variations, the volume of the liquid formulations described herein administered subconjunctivally to a rabbit eye or a subject's eye, including but not limited to a human subject's eye, is between about 0.1 μl and about 200 μl, between about 50 μl and about 200 μl, between about 200 μl and about 300 μl, between about 300 μl and about 400 μl, between about 400 μl and about 500 μl, between about 600 μl and about 700 μl, between about 700 μl and about 800 μl, between about 800 μl and about 900 μl, between about 900 μl and about 1000 μl, between about 50 μl and about 150 μl, between about 0.1 μl and about 10 ... In some variations, the volume of the liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 10 μl. In some variations, the volume of the liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 5 μl. In some variations, the volume of the liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 5 μl. In some variations, the volume of the liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye (including but not limited to a human subject's eye) is between about 1 μl and about 5 μl. In some variations, the volume of a liquid formulation described herein administered subconjunctivally to a rabbit eye or a subject's eye, including but not limited to a human subject's eye, is between about 0.1 μl and about 200 μl.

In some variations, the liquid formulations described herein are administered to multiple subconjunctival locations over a period of time (including, but not limited to, within one hour of each other). Without being bound by theory, it is believed that such multiple administrations (e.g., multiple injections) allow for a larger total dose to be administered subconjunctivally compared to a single dose because the ability of local ocular tissue to absorb larger volumes may be limited.

One liquid formulation described herein is an in situ gelling formulation. The in situ gelling formulations described herein comprise a therapeutic agent and a plurality of polymers, which provide a formulation that forms a gel or gel-like substance when placed in an aqueous medium, including but not limited to an aqueous medium of the eye.

In some variations of the liquid formulations described herein, the therapeutic agent is a solution or suspension of rapamycin in a liquid medium. The liquid medium includes, but is not limited to, a solvent, including, but not limited to, the solvent described in the "therapeutic agent solubilization" section.

Liquid formulations described herein may include a solubilizing agent component. In some variations, the solubilizing agent component is a surfactant. Note that there is overlap between components that can be solvents and solubilizing agents, so the same component can be used as a solvent or solubilizing agent in some systems. Liquid formulations contain a therapeutic agent and a component that can be considered a solvent, a solubilizing agent, or a surfactant if the component acts as a solvent; or a solubilizing agent or a surfactant if the component does not act as a solvent.

The liquid formulation may also optionally contain stabilizers, excipients, gelling agents, adjuvants, antioxidants, and/or other ingredients described herein.

In some variations, all ingredients of the liquid formulation except the therapeutic agent are liquid at room temperature.

In some variations, the liquid formulation comprises a release modifier. In some variations, the release modifier is a film-forming polymer component. The film-forming polymer component may comprise one or more film-forming polymers. Any film-forming polymer may be used in the excipient component. In some variations, the film-forming polymer component comprises a polymer that forms a water-insoluble film. In some variations, the release modifier component comprises an acrylate polymer, including but not limited to polymethacrylates, including but not limited to Eudragit RL.

Described herein are compositions and liquid formulations for delivering the therapeutic agents described in the "Therapeutic Agents" section. Delivery of therapeutic agents using the compositions and liquid formulations described herein can be used to treat, prevent, inhibit, delay the onset of, or cause regression of the diseases and conditions described in the "Diseases and Conditions" section. The compositions and liquid formulations described herein can contain any of the therapeutic agents described in the "Therapeutic Agents" section, including but not limited to rapamycin. The compositions and liquid formulations described herein can contain one or more than one therapeutic agent. Compositions and liquid formulations other than those specifically described herein can be used.

When therapeutic agent is rapamycin, composition and liquid preparation can be used for keeping a certain amount of rapamycin in the vitreous, and described amount can effectively treat wet AMD. In a non-limiting example, it is believed that the liquid preparation of following delivery rapamycin can be used for treating wet AMD, and described preparation can maintain the rapamycin concentration of about 10pg/ml to about 2 μ g/ml in the vitreous for a period of time. When rapamycin is contained in the liquid preparation that forms non-dispersed agglomerates, the rapamycin concentration stated represents the amount that effectively treats eye disease or illness, rather than being merely present in the form of non-dispersed agglomerates. In another non-limiting example, it is believed that the delivery system of following delivery rapamycin can be used for treating wet AMD, and described delivery system can maintain the rapamycin concentration of about 0.01pg/mg to about 10ng/mg in retinal choroidal tissue for a period of time. The therapeutic agent of other therapeutically effective amounts is also possible, and can be easily determined by those skilled in the art according to the teachings of this paper.

When the therapeutic agent is rapamycin, the compositions and liquid formulations described herein can be used to deliver a dose of rapamycin to a subject (including but not limited to a human subject) or to the subject's eye. In one non-limiting example, it is believed that a liquid formulation containing a dose of about 20 μg to about 4 mg can be used to treat wet AMD.

In some variations, the therapeutic agent in the liquid formulation comprises between about 0.01% and about 30%; between about 0.05% and about 15%; between about 0.1% and about 10%; between about 1% and about 5%; or between about 5% and about 15%; between about 8% and about 10%; between about 0.01% and about between about 1% and about 5%; between about 5% and about 10%; between about 15% and about 30%, between about 20% and about 30%; or between about 25% and about 30%.

A person skilled in the art can determine what amount or concentration of a given therapeutic agent is equivalent to a certain amount or concentration of rapamycin based on the teachings herein, for example, by administering the therapeutic agent in different amounts or concentrations to a disease model system (e.g., an in vivo or in vitro model system) and comparing the results in the model system relative to the results of various amounts or concentrations of rapamycin. A person skilled in the art can also determine what amount or concentration of a given therapeutic agent is equivalent to a certain amount or concentration of rapamycin based on the teachings herein by reviewing experiments comparing rapamycin with other therapeutic agents in the scientific literature. It should be understood that even the same therapeutic agent can have different levels of rapamycin equivalence when, for example, different diseases or conditions are being evaluated, or different types of formulations are being used. Non-limiting examples of scientific literature comparing rapamycin with other therapeutic agents for ocular diseases are Ohia et al., Effects of steroids and immunosuppressive drugs on endotoxin-uveitis in rabbits, J. Ocul. Pharmacol. 8(4):295-307 (1992); Kulkarni, Steroida and nonsteroidal drugs in endotoxin-induced uveitis, J. Ocul. Pharmacol. 10(1):329-34 (1994); Hafizi et al., Differential effects of rapamycin, cyclosporine A, and FK506 on human coronary artery smooth muscle cell proliferation and signaling, Vascul Pharmacol. 41(4-5):167-76 (2004); and US 2005/0187241.

For example, in a wet AMD model, if a therapeutic agent is found to be 10-fold less potent or effective than rapamycin in treating wet AMD, a therapeutic agent concentration of 10 ng/ml should be equivalent to a rapamycin concentration of 1 ng/ml. Alternatively, if a therapeutic agent is found to be 10-fold less potent or effective than rapamycin in treating wet AMD, a 10-fold amount of the therapeutic agent relative to the amount of rapamycin should be administered.

The solvent component may, for example, constitute between about 0.01% and about 99.9%; between about 0.1% and about 99%; between about 25% and about 55%; between about 30% and about 50%; or between about 35% and about 45%; between about 0.1% and about 10%; between about 10% and about 20%; between about 20% and about 30%; between about 30% and about 40%; between about 40% and about 45%; between about 40% and about 45%; between about 45% and about 50%; between about 50% and about 60%; between about 50% and about 70%; between about 70% and about 80%; between about 80% and about 90%; or between about 90% and about 100%, of the total weight of the composition.

The solubilizer component may, for example, comprise between about 0.01% and about 30%; between about 0.1% and about 20%; between about 2.5% and about 15%; between about 10% and about 15%; or between about 5% and about 10%; between about 8% and about 12%; between about 10% and about 20%; or between about 20% and about 30% of the total weight of the composition.

In some variations, the liquid formulations described herein have a viscosity between 40% and 120% centipoise. In some variations, the liquid formulations described herein have a viscosity between 60% and 80% centipoise.

In some variations, the liquid formulations described herein contain a therapeutic agent and a solvent component. The solvent component may contain a single solvent or a combination of solvents. The therapeutic agent component may contain a single therapeutic agent or a combination of therapeutic agents. In some variations, the solvent is glycerol, dimethyl sulfoxide, N-methyl pyrrolidone, dimethylacetamide (DMA), dimethylformamide, glycerol formal, ethoxydiglycol, triethylene glycol dimethyl ether, triacetin, glyceryl diacetate, corn oil, acetyl triethyl citrate (ATC), ethyl lactate, PEGylated caprylic acid glyceride, gamma butyrolactone, dimethyl isosorbide dimethyl ester, benzyl alcohol, ethanol, isopropyl alcohol, polyethylene glycols of various molecular weights (including but not limited to PEG300 and PEG400) or propylene glycol, or a mixture of one or more thereof.

In some variations, the liquid formulations described herein are solutions and comprise a therapeutic agent and a solvent component. In some variations, the solvent component comprises ethanol. In some variations, the solvent component comprises ethanol and polyethylene glycol, including but not limited to liquid polyethylene glycol (including but not limited to one or more of PEG 300 or PEG 400).

In some variations, the liquid formulations described herein contain no more than about 250 μl of polyethylene glycol. In some variations, the liquid formulations described herein contain no more than about 250 μl, no more than about 200 μl, no more than about 150 μl, no more than about 125 μl, no more than about 100 μl, no more than about 75 μl, no more than about 50 μl, no more than about 25 μl, no more than about 20 μl, no more than about 15 μl, no more than about 10 μl, no more than about 7.5 μl, no more than about 5 μl, no more than about 2.5 μl, no more than about 1.0 μl, or no more than about 0.5 μl of polyethylene glycol. Preparations containing polyethylene glycol can contain, for example, PEG300 or PEG400.

In some variations, the liquid formulations described herein are suspensions and comprise a therapeutic agent and a diluent component. In some variations, the diluent component contains one or more ingredients listed herein as solvents or solubilizers, wherein the resulting mixture is a suspension.

In some variations, the liquid formulation is part solution and part suspension.

In some variations, the liquid formulation is an in situ gelling formulation and comprises a therapeutic agent and a polymer component, wherein the polymer component can comprise a plurality of polymers. In some variations, the liquid formulation comprises a polymethacrylate polymer. In some variations, the liquid formulation comprises a polyvinylpyrrolidone polymer.

Some variations of the liquid formulation contain between about 0.01% and about 20% by weight of a therapeutic agent (such as, but not limited to, rapamycin), between about 5% and about 15% by weight of a solvent, between about 5% and about 15% by weight of a solubilizing agent (including, but not limited to, a surfactant), and water as the primary retaining ingredient. In some variations, the formulation further comprises between about 0% and about 40% by weight of a stabilizer, excipient, adjuvant, or antioxidant.

In some variations, the liquid formulation comprises up to about 5% by weight of the therapeutic agent (including but not limited to rapamycin); and up to about 99.9% by weight of the solvent component. In some variations, the liquid formulation comprises up to about 5% by weight of the therapeutic agent (including but not limited to rapamycin); and up to about 99.9% by weight of the diluent component.

In some variations, the liquid formulation comprises up to about 5% by weight of a therapeutic agent (including but not limited to rapamycin); up to about 10% by weight of a solvent component; and up to about 85% by weight of a solubilizing component. In some variations, the solubilizing component is an aqueous solution of a surfactant.

The various polymeric components may, for example, comprise between about 0.01% and about 30%; between about 0.1% and about 20%; between about 2.5% and about 15%; between about 10% and about 15%; between about 3% and about 5%; between about 5% and about 10%; between about 8% and about 12%; between about 10% and about 20%; or between about 20% and about 30% of the total weight of the composition.

Some variations of the liquid formulations contain between about 0.01% and about 20% by weight of a therapeutic agent (including, but not limited to, rapamycin), between about 60% and about 98% by weight of a solvent component, and a plurality of polymers, the combined percentages of which are between about 0.1% and about 15% by weight of the total weight. In some variations, the formulations further contain between about 0% and about 40% by weight of a stabilizer, excipient, adjuvant, or antioxidant.

In some variations, the liquid formulation may contain about 4% by weight of the therapeutic agent (including but not limited to rapamycin); about 91% by weight of the solvent; and about 5% by weight of the polymer component.

Some examples and variations of the liquid formulations described herein were prepared and listed in Table 1. The listed formulations are referred to as one or more solutions ("S"), suspensions ("SP"), emulsions ("E"), or in situ gels ("ISG") according to their type. Some suspensions list the median particle size. As described herein, some liquid formulations form non-dispersed clumps after, for example, injection into an aqueous environment (such as the vitreous humor of the eye). For these formulations injected into the vitreous humor of a rabbit eye, the right-hand column of Table 1 indicates whether a non-dispersed clump (NDM) is formed after a particular volume is injected into the vitreous humor of a rabbit eye.

The following references show one or more formulations (including but not limited to rapamycin formulations), are incorporated herein in their entirety, and describe the use of various doses of rapamycin and other therapeutic agents for treating a variety of diseases or conditions: US 60/651,790, filed February 9, 2005, entitled FORMULATIONS FOR OCULAR TREATMENT, attorney docket number 57796-30002.00; US 60/664,040, filed February 9, 2005, attorney docket number 57796-30004.00, entitled LIQUID FORMULATIONS FOR TREATMENT OF DISEASES OR CONDITIONS; US 60/664,119, filed March 21, 2005, attorney docket number 57796-30005.00, entitled DRUG DELIVERY SYSTEMS FOR TREATMENT OF DISEASES OR CONDITIONS. CONDITIONS; US60/664,306, filed 3/21/2005, attorney docket number 57796-30006.00, entitled IN SITUGELLING FORMULATIONS AND LIQUID FORMULATIONS FOR TREATMENT OF DISEASES OR CONDITIONS; US_/_, filed 2/9/2006, entitled FORMULATIONS FOR OCULAR TREATMENT, attorney docket number 57796-20002.00; _/_, filed 2/9/2006, attorney docket number 57796-20004.00, entitled LIQUID FORMULATIONS FOR TREATMENT OF DISEASES OR CONDITIONS; US2005/0187241 and US2005/0064010.

Liquid formulations that form non-dispersible clumps

A class of liquid preparations described herein forms non-dispersed clumps when placed in an aqueous medium. "Non-dispersed clumps" is used herein to refer to the structure or shape formed relative to the environment in which the liquid preparation is placed when the liquid preparation is placed in the environment. Generally speaking, the non-dispersed clumps of a liquid preparation are any situation except that the liquid preparation is uniformly distributed in the surrounding medium. Non-dispersed clumps can be pointed out, for example, by observing the liquid preparation being applied and characterizing its outward appearance relative to the surrounding environment.

In some variations, the aqueous medium is water. In some variations, the aqueous medium is deionized water, distilled water, sterile water, or tap water (including but not limited to tap water available within the purview of MacuSight in Union City, California).

In some variations, the aqueous medium is an aqueous medium of a subject. In some variations, the aqueous medium is an aqueous medium of a subject's eye, including but not limited to the vitreous humor of a subject's eye. In some variations, the subject is a human subject. In some variations, the subject is a rabbit.

In some variations, the liquid formulation forms non-dispersible clumps when exposed to a temperature or temperature range, including but not limited to about room temperature, about ambient temperature, about 30°C, about 37°C, or about the temperature of the subject's aqueous medium.

In some variations, the liquid formulation forms non-dispersible clumps when exposed to a pH or pH range, including but not limited to a pH between about 6 and about 8.

In some variations, the non-dispersed mass comprises a gel or gel-like substance.

In some variations, the non-dispersed mass comprises a polymer matrix. In some variations, the non-dispersed mass comprises a polymer matrix in which the therapeutic agent is dispersed.

Liquid preparation described herein can be generally any geometric shape or shape after being applied to experimenter or experimenter's eye (including but not limited to human experimenter).In some modifications, non-dispersed agglomerate is between about 0.1mm and about 5mm.In some modifications, non-dispersed agglomerate is between about 1mm and about 3mm.When the liquid preparation that forms non-dispersed agglomerate is applied to vitreous body, can be shown as for example dense spherical agglomerate.In some cases, liquid preparation can be presented as non-dispersed agglomerate with respect to surrounding medium, wherein said non-dispersed agglomerate definition is more unclear, and its geometric shape is more amorphous compared with spherical.

The liquid preparation of the non-dispersed agglomerate of formation described herein can form non-dispersed agglomerate when being placed in medium at once, or non-dispersed agglomerate can form after placing liquid preparation for a period of time.In some modifications, non-dispersed agglomerate formed in approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days or approximately 7 days.In some modifications, non-dispersed agglomerate formed in approximately 1 week, approximately 2 weeks or approximately 3 weeks.

In some variations, the liquid formulations described herein that form a non-dispersible mass appear as a milky or whitish semi-continuous or semi-solid non-dispersible mass relative to the medium in which it is placed.

A kind of liquid preparation as described herein forms following non-dispersed mass, and the non-dispersed mass forms solid depot when preparation is injected into any or all water (between rabbit eye vitreous or rabbit eye sclera and conjunctiva).A kind of liquid preparation as described herein forms following non-dispersed mass, and the non-dispersed mass forms semisolid when preparation is injected into any or all water (between rabbit eye vitreous or rabbit eye sclera and conjunctiva).A kind of liquid preparation as described herein forms following non-dispersed mass, and the non-dispersed mass forms polymer matrix when preparation is injected into any or all water (between rabbit eye vitreous or rabbit eye sclera and conjunctiva).A kind of liquid preparation as described herein forms following non-dispersed mass, and the non-dispersed mass has the form of gel, hydrogel or gel-like material when preparation is injected into any or all water (between rabbit eye vitreous or rabbit eye sclera and conjunctiva).

In some variations described herein, when the surrounding medium is aqueous, the liquid formulation forms a non-dispersed mass relative to the surrounding medium. An "aqueous medium" or "aqueous environment" is a medium or environment that contains at least about 50% water. Examples of aqueous media include, but are not limited to, water, vitreous humor, extracellular fluid, conjunctiva, sclera, the space between the sclera and conjunctiva, aqueous humor, gastric fluid, and any tissue or body fluid that contains at least about 50% water. Aqueous media include, but are not limited to, gel structures, including, but not limited to, gel structures of the conjunctiva and sclera.

In some variations, the liquid formulations described herein form non-dispersed clumps when a test volume of the liquid formulation is placed in the vitreous of a rabbit eye. In some variations, the test volume is administered to the rabbit eye, and the test volume is equal to the volume of the liquid formulation administered to a subject, including but not limited to the eye of a human subject.

In some variations, the test volume administered to the rabbit eye is equal to the volume administered to the subject's eye multiplied by a scaling factor, and the scaling factor is equal to the average volume of the rabbit eye divided by the average volume of the subject's eye. As used herein, the "average volume" of an eye generally refers to the average volume of the eyes of similarly aged members of the species under consideration, rather than the average volume of any particular individual's eye.

In some variations, the test volume administered to the rabbit eye is between about 10 μl and about 50 μl. In some variations, the test volume administered to the rabbit eye is between about 1 μl and about 30 μl. In some variations, the test volume administered to the rabbit eye is between about 50 μl and about 100 μl. In some variations, the test volume administered to the rabbit eye is between about 25 μl and about 75 μl. In some variations, the test volume administered to the rabbit eye is about 30 μl.

In some variations, a liquid formulation that forms non-dispersible clumps when placed in a medium comprises one or more therapeutic agents at a concentration between about 0.01% and about 10% by weight, and a solvent at a concentration between about 10% and about 99% by weight. In some variations, the liquid formulation further comprises a solubilizing agent, including but not limited to a surfactant. In some variations, the liquid formulation further comprises a stabilizer, excipient, adjuvant, or antioxidant, etc., at a concentration between about 0% and about 40% by weight. In some variations, the therapeutic agent comprises about 5% by weight, and the solvent component comprises about 95% by weight.

Whether a liquid formulation exhibits non-dispersed clumps relative to the surrounding medium when present in a subject (including but not limited to a human subject) or in the eye of a subject can be determined by mixing the therapeutic agent with a solvent, administering to the vitreous of the eye of a subject (including but not limited to a human subject), and comparing the liquid formulation to the surrounding medium.

A liquid formulation that can be used to treat, prevent, inhibit, delay the occurrence of, or resolve a disease or condition in a subject (including but not limited to a human subject) is a liquid formulation that forms non-dispersed clumps when placed in the vitreous body of a rabbit eye. When used to treat, prevent, inhibit, delay the occurrence of, or resolve a disease or condition in a subject, the liquid formulation is administered to the subject. The liquid formulation may or may not form non-dispersed clumps in the subject. A liquid formulation described herein forms non-dispersed clumps when administered to a subject, and forms non-dispersed clumps when administered to a rabbit eye.

Without being bound by theory, it is believed that the low solubility of rapamycin in the vitreous contributes to the formation of non-dispersed clumps in some of the rapamycin-containing liquid formulations described herein. The vitreous is a clear gel composed essentially entirely of water (up to 99%). Without being bound by theory, it is believed that when the rapamycin in the injected formulation comes into contact with the vitreous, precipitation of the rapamycin occurs.

Without being bound by theory, it is believed that factors that influence the formation and shape of non-dispersed clumps include the concentration of rapamycin in the formulation, the viscosity of the formulation, the ethanol content of the formulation, and the injection volume. It is believed that maintaining a higher concentration of rapamycin after injection of the formulation promotes the formation of non-dispersed clumps, as opposed to lower local concentrations of rapamycin after injection of the formulation. As the volume of a given dose increases, the formation of non-dispersed clumps is less favored. When the rapamycin concentration is increased and/or the viscosity is increased, the formation of non-dispersed clumps may be more favored. The ethanol content affects the solubility of rapamycin in the formulation and the viscosity of the formulation.

In one comparison, 100 μl of a solution of 0.4% rapamycin, 4.0% ethanol, and 95.6% PEG400 (a 400 μg dose) did not form non-dispersed clumps after injection into rabbit eyes. In contrast, 20 μl of a solution of 2.00% rapamycin, 4.0% ethanol, and 94% PEG400 (also a 400 μg dose) formed dense, spherical, non-dispersed clumps after injection into rabbit eyes.

Without being bound by theory, in the latter example, it is hypothesized that the formation of the non-dispersed mass occurs as described in Figures 1A-1C and below. Upon injection, the liquid formulation, due to its viscosity, forms a spherical mass 100 in the vitreous humor 110. Ethanol then diffuses from the spherical mass, forming a localized precipitation of rapamycin within the spherical mass 120. Finally, polyethylene glycol also diffuses from the spherical mass, resulting in a solid, dense, non-dispersed mass 130 of rapamycin.

In some variations, the non-dispersed mass described herein is comprised of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% by volume of the therapeutic agent when injected into the vitreous of a rabbit eye.

In some variations, upon forming a non-dispersed mass containing rapamycin, delivery is sustained at a substantially constant rate over an extended period of time. Without being bound by any theory, it is believed that delivery of rapamycin from the non-dispersed mass within the vitreous is dependent upon dissolution of the rapamycin within the vitreous, which in turn is dependent upon clearance of the drug from the vitreous to other tissues. Without being bound by any theory, it is believed that this release process maintains a steady-state concentration of rapamycin within the vitreous.

In some variations, the formation of a non-dispersed bolus reduces the toxicity of the injected liquid formulation compared to an equivalent dose that does not form a non-dispersed bolus. In variations where the liquid formulation injected into the vitreous does not form a non-dispersed bolus, the drug (e.g., rapamycin) has been shown to disperse within the vitreous. In some variations, this may interfere with vision.

In some variations, the liquid formulation, which is a suspension, forms a non-dispersed mass upon injection into the vitreous. The formation of a non-dispersed mass from an injected suspension may be more advantageous as the suspension particle size increases.

In some variations, the liquid formulation is believed to form a visually observable, non-dispersed mass upon injection into the eye of a subject, including but not limited to a human subject.

In some variations, it is believed that the liquid formulation forms a non-dispersed mass upon subconjunctival injection. In some variations, it is believed that subconjunctival administration of the liquid formulation forms a reservoir in the scleral tissue. That is, it is believed that the therapeutic agent is absorbed into the sclera proximal to the injection site and forms a local concentration of the drug in the sclera.

In situ gelling preparations

Described herein are liquid formulations that form a non-dispersible mass that forms a gel or gel-like substance when placed in an aqueous medium. In some variations, the non-dispersible mass comprises a gel; in some variations, the gel is a hydrogel.

As used herein, "in situ gelling formulation" refers to a liquid formulation that forms a gel-like, non-dispersible mass when placed in an aqueous medium (including but not limited to water, the vitreous humor of a rabbit eye, and the aqueous medium between the sclera and conjunctiva of a rabbit eye). In some variations, the in situ gelling formulation forms a gel-like, non-dispersible mass when placed in tap water.

In some variations, the in situ gelling preparation is a suspension before being placed in an aqueous medium, and forms a gel when placed in an aqueous medium. In some variations, the in situ gelling preparation is a solution before being placed in an aqueous medium, and forms a gel in situ when placed in an aqueous medium. In some variations, the in situ gelling preparation is an emulsion before being placed in an aqueous medium, and forms a gel when placed in an aqueous medium. In some variations, the in situ gelling preparation is placed in an aqueous medium (including but not limited to any one or all of water, vitreous body, or sclera and conjunctiva) to form a gel-like non-dispersed mass. In some variations, the in situ gel is formed by a polymer matrix. In some variations, the therapeutic agent is dispersed in the polymer matrix.

Described herein are in situ gelling formulations that can be used to treat, prevent, inhibit, delay the onset of, or cause regression of a disease or condition in a subject, including but not limited to a human subject. When used to treat, prevent, inhibit, delay the onset of, or cause regression of a disease or condition in a subject, the in situ gelling formulation is administered to the subject. A liquid formulation described herein comprises an in situ gelling formulation that forms a non-dispersed mass when administered to a subject and forms a non-dispersed mass when administered to a rabbit eye.

In some variations, the in situ gelling formulation comprises one or more polymers. Various types of polymers are described herein, including polymers that are solvents, polymers that are solubilizers, polymers that are release modifiers, polymers that are stabilizers, and the like. In some variations, any combination of polymers is used, wherein when placed in an aqueous medium (including but not limited to water, vitreous humor, or any or all of the space between the sclera and conjunctiva), the polymers, when combined with the therapeutic agent, form any or all of a non-dispersed mass, gel, hydrogel, or polymer matrix.

In some variations, the in situ gelling formulation, when administered to a subject, delivers extended release of the therapeutic agent to the subject.

In some variations, the liquid formulation comprises a therapeutic agent and a plurality of polymers, one of which is a polymethacrylate. Polymethacrylates are known by various names and are available in a variety of formulations, including, but not limited to, polymethacrylate, methacrylic acid-ethyl acrylate copolymer (1:1), methacrylic acid-ethyl acrylate copolymer (1:1) with a dispersion of 30%, methacrylic acid-methyl methacrylate copolymer (1:2), methacrylic acid-methyl methacrylate copolymer (1:1), acidum methacrylicum et ethylis acrylas polymerisatum 1:1, acidum methacrylicum et ethylis methacrylas polymerisatum 1:1 dispersion 30 percent, acidum methacrylicum et methylis methacrylas polymerisatum 1:1, acidum methacrylicumet methylis methacrylas polymerisatum 1:2, USPNF: ammonium methacrylate copolymer, methacrylic acid copolymer, methacrylic acid copolymer dispersion.

In some variations, one of the polymers is polyvinylpyrrolidone. Polyvinylpyrrolidone is known by various names and is available in a variety of formulations, including, but not limited to, povidone, povidonum, kollidon; plasdone; poly[1-(2-oxo-1-pyrrolidinyl)ethylene]; polyvidone; PVP; 1-vinyl-2-pyrrolidinyl polymer and 1-vinyl-2-pyrrolidone homopolymer.

A liquid formulation described herein comprises a therapeutic agent and a solvent component. The solvent component can comprise a single solvent or a combination of solvents.

In some variations, the solvent is glycerol, dimethyl sulfoxide, N-methylpyrrolidone, ethanol, isopropyl alcohol, polyethylene glycols of various molecular weights (including but not limited to PEG 300 and PEG 400), or propylene glycol, or a mixture of one or more thereof.

In some variations, the solvent is polyethylene glycol. Polyethylene glycol is known by various names and is available in a variety of formulations, including, but not limited to, polyethylene glycol, polyethylene glycol 400, polyethylene glycol 1500, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 20000, macrogola, breox PEG; carbowax; carbowax sentry; Hodag PEG; Lipo; Lipoxol; Lutrol E; PEG; Pluriol E; polyethylene glycol and α-hydro-ω-hydroxy-poly(oxy-1,2-ethanediyl).

Compositions and liquid formulations for delivering therapeutic agents

The compositions and liquid formulations described herein can be used to deliver an amount of therapeutic agent that is effective to treat, prevent, inhibit, delay the onset of, or cause regression of the diseases and conditions described in the "Diseases and Conditions" section. In some variations, the compositions and liquid formulations described herein deliver one or more therapeutic agents over an extended period of time.

An "effective amount" of a therapeutic agent for administration as described herein (which is also referred to herein as a "therapeutically effective amount") refers to the amount of the therapeutic agent that is sought to provide a therapeutic effect when administered to a subject (including but not limited to a human subject). Achieving different therapeutic effects may require different effective amounts of the therapeutic agent. For example, a therapeutically effective amount of a therapeutic agent for preventing a disease or condition may be different from a therapeutically effective amount for treating, inhibiting, delaying the onset of, or causing the regression of a disease or condition. In addition, the therapeutically effective amount may depend on the subject's age, weight, and other health conditions known to those skilled in the art of treating the disease or condition. Thus, the therapeutically effective amount may be different in each subject to which the therapeutic agent is administered.

An effective amount of a therapeutic agent for treating, preventing, inhibiting, delaying the onset of, or causing regression of a particular disease or condition is also referred to herein as an amount of the therapeutic agent effective to treat, prevent, inhibit, delay the onset of, or cause regression of the disease or condition.

To determine whether the level of a therapeutic agent is a "therapeutically effective amount" for treating, preventing, inhibiting, delaying the onset of, or causing regression of the diseases and conditions described in the "Diseases and Conditions" section, the liquid formulation can be administered to an animal model of the disease or condition of interest and the effects observed. Additionally, dosages within the range of human clinical trials can be conducted to determine a therapeutically effective amount of the therapeutic agent.

Generally, the therapeutic agent can be formulated in any composition or liquid formulation that is capable of delivering a therapeutically effective amount of the therapeutic agent to a subject or an eye of a subject over a desired delivery time. Compositions include liquid formulations.

Solubilization of therapeutic agents

One composition or liquid formulation that can be used is one in which the therapeutic agent is dissolved in a solvent component. Generally, any solvent having the desired effect can be used in which the therapeutic agent is dissolved. In some variations, the solvent is aqueous. In some variations, the solvent is non-aqueous. An "aqueous solvent" is a solvent that contains at least about 50% water.

In general, any concentration of the dissolved therapeutic agent having the desired effect can be used. The solvent component can be a single solvent or a mixture of solvents. The types of solvents and solutions are well known to those skilled in the art of drug delivery technology. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott Williams &Wilkins; 20th edition (December 15, 2000); Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th edition, Lippincott Williams & Wilkins (August 2004); Handbook Of Pharmaceutical Excipients 2003, American Pharmaceutical Association, Washington, DC, USA and Pharmaceutical Press, London, UK; and Strickley, Solubilizing Excipients in Oral and Injectable Formulations, Pharmaceutical Research, Vol. 21, No. 2, February 2004.

As previously mentioned, some solvents may also act as solubilizing agents.

Solvents that can be used include, but are not limited to, DMSO, ethanol, methanol, isopropanol; castor oil, propylene glycol, glycerol, polysorbate 80, benzyl alcohol, dimethylacetamide (DMA), dimethylformamide (DMF), triacetin, diacetin, corn oil, acetyl triethyl citrate (ATC), ethyl lactate, glycerol formal, ethoxydiglycol (Transcutol, Gattefosse), triglyme, dimethyl isosorbide (DMI), gamma-butyrolactone, N-methyl-2-pyrrolidone (NMP), polyethylene glycols of various molecular weights (including, but not limited to, PEG300 and PEG400), and PEGylated caprylic glyceride (Labrasol, Gattefosse), any one or more combinations of the foregoing, or any one or more analogs or derivatives of the foregoing.

In some variations, the solvent is polyethylene glycol. Polyethylene glycol is known by various names and is available in a variety of formulations, including, but not limited to, polyethylene glycol, polyethylene glycol 400, polyethylene glycol 1500, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 20000, macrogola, breox PEG; carbowax; carbowax sentry; Hodag PEG; Lipo; Lipoxol; Lutrol E; PEG; Pluriol E; polyethylene glycol and α-hydro-ω-hydroxy-poly(oxy-1,2-ethanediyl).

In some variations, the polyethylene glycol is liquid PEG, and is one or more of PEG300 or PEG400.

Other solvents include a _C6 - _C24 fatty acid in an amount sufficient to solubilize the therapeutic agent.

Phospholipid solvents may also be used, such as lecithin, phosphatidylcholine, or a mixture of various diglycerides (diglycerides of stearic acid, palmitic acid, and oleic acid) linked to a choline phosphate ester; hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG), L-α-dimyristoylphosphatidylcholine (DMPC), L-α-dimyristoylphosphatidylglycerol (DMPG).

Other examples of solvents include, for example, alcohols, propylene glycol, polyethylene glycols of varying molecular weights, propylene glycol esters, propylene glycol esterified with fatty acids such as oleic acid, stearic acid, palmitic acid, capric acid, and linoleic acid; medium-chain mono-, di-, or triglycerides; long-chain fatty acids; naturally occurring oils; and mixtures thereof. The oil component of the solvent system includes commercially available oils and naturally occurring oils. The oil may also be a vegetable oil or a mineral oil. The oil may be characterized as a non-surface-active oil, typically lacking a hydrophilic-lipophilic balance. Commercially available materials containing heavy-chain triglycerides include, but are not limited to, Captex 100, Captex 300, Captex 355, Miglyol 810, Miglyol 812, Miglyol 818, Miglyol 829, and Dynacerin 660. Commercially available propylene glycol ester compositions include Captex 200 and Miglyol 840, among others. The commercially available product, Capmul MCM, contains one of many possible medium-chain mixtures containing mono- and diglycerides.

Other solvents include naturally occurring oils such as peppermint oil and seed oils. Exemplary natural oils include oleic acid, castor oil, safflower seed oil, soybean oil, olive oil, sunflower oil, sesame oil, and peanut oil. Soybean fatty acids may also be used. Examples of fully saturated non-aqueous solvents include, but are not limited to, esters of medium to long-chain fatty acids (e.g., fatty acid triglycerides with chain lengths of about C ₆ to about C ₂₄ ). Hydrogenated soybean oil and other vegetable oils may also be used. Fatty acid mixtures may be separated and refined from natural oils (e.g., coconut oil, palm kernel oil, Brazilian palm oil, etc.). In some embodiments, medium-chain (about C ₈ to about C ₁₂ ) triglycerides such as caprylic/capric triglycerides from coconut oil or palm kernel oil may be used. Medium-chain monoglycerides or diglycerides may also be used. The non-aqueous solvent of claim 1 is saturated.Other fully saturated non-aqueous solvents include, but are not limited to, saturated coconut oil (which generally includes a mixture of lauric acid, myristic acid, palmitic acid, capric acid and caproic acid), including those sold under the trade name Miglyol ^™ from Huls and having trade names 810, 812, 829 and 840. The NeoBee ^™ product sold by Drew Chemicals is also mentioned.Non-aqueous solvents include isopropyl myristate. Examples of synthetic oils include triglycerides and propylene glycol esters with saturated or unsaturated fatty acids having 6 to 24 carbon atoms, such as caproic acid, octanoic acid (caprylic acid), pelargonic acid (pelargonic acid), capric acid (capric acid), undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid (myristic acid), pentadecanoic acid, hexadecanoic acid (palmitic acid), heptadecanoic acid, octadecanoic acid (stearic acid), nonadecanoic acid, heptadecanoic acid, eicosanoic acid, unicosanoic acid, behenic acid, and tetracosanoic acid etc. Examples of unsaturated carboxylic acids include oleic acid, linoleic acid, and linolenic acid. The non-aqueous solvent may include monoglycerides, diglycerides, and triglycerides of fatty acids or mixed glycerides and/or propylene glycol monoesters or diesters, wherein at least one glycerol molecule is esterified with fatty acids having different carbon atom lengths. A non-limiting example of a "non-oil" used as a solvent is polyethylene glycol.

Exemplary vegetable oils include cottonseed oil, corn oil, sesame oil, soybean oil, olive oil, fractionated coconut oil, peanut oil, sunflower oil, safflower oil, almond oil, avocado oil, palm oil, palm kernel oil, babassu oil, beechnut oil, linseed oil, rapeseed oil, etc. Monoglycerides, diglycerides, and triglycerides of vegetable (including but not limited to corn) oils can also be used.

Cross-linked or uncross-linked polyvinylpyrrolidone (PVP) can also be used as a solvent. Other solvents include, but are not limited to, _C6 - _C24 fatty acids, oleic acid, Imwitor 742, Capmul, F68, F68 (Lutrol), PLURONICS (including, but not limited to, PLURONICS F108, F127, and F68, poloxamers, Jeffamines), Tetronics, F127; cyclodextrins such as α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin (Captisol); CMC, polysorbitan 20, Cavitron, and polyethylene glycols of various molecular weights, including, but not limited to, PEG 300 and PEG 400.

Beeswax and d-α-tocopherol (vitamin E) may also be used as solvents.

Solvents for liquid formulations can be determined by a variety of methods known in the art, including but not limited to (1) theoretically estimating their solubility parameter values using standard equations in the art and selecting a solvent that matches the therapeutic agent; and (2) experimentally determining the saturated solubility of the therapeutic agent in the solvent and selecting a solvent that exhibits the desired solubility.

Solubilization of rapamycin

When the therapeutic agent is rapamycin, solvents that can be used to prepare rapamycin solutions or suspensions include, but are not limited to, any of the solvents described herein, including, but not limited to, DMSO, glycerol, ethanol, methanol, isopropanol; castor oil, propylene glycol, polyethylene propylene, glycerol, polysorbate 80, benzyl alcohol, dimethylacetamide (DMA), dimethylformamide (DMF), glycerol formal, ethoxydiglycol (Transcutol, Gattefosse), triglyme, dimethyl isosorbide (DMI), gamma-butyrolactone, N-methyl-2-pyrrolidone (NMP), polyethylene glycols of various molecular weights (including, but not limited to, PEG300 and PEG400), and any one or more of PEGylated caprylic glyceride (Labrasol, Gattefosse).

Other solvents include, but are not limited to, _C6 - _C24 fatty acids, oleic acid, Imwitor 742, Capmul, F68, F68 (Lutrol), PLURONICS (including, but not limited to, PLURONICS F108, F127, and F68, poloxamers, Jeffamines), Tetronics, F127, β-cyclodextrin, CMC, polysorbitan 20, Cavitron, softigen 767, captisol, and sesame oil.

Other methods that can be used to solubilize rapamycin are described in Solubilization of Rapamycin, P. Simamora et al. Int'l J. Pharma 213 (2001) 25-29, the entire contents of which are incorporated herein by reference.

As a non-limiting example, rapamycin can be dissolved in 5% DMSO or methanol in a balanced salt solution. The rapamycin solution can be an unsaturated, saturated, or supersaturated rapamycin solution. The rapamycin solution can be contacted with solid rapamycin. In a non-limiting example, rapamycin can be dissolved to a concentration of up to about 400 mg/ml. Rapamycin can also be dissolved, for example, in propylene glycol esterified with fatty acids such as oleic acid, stearic acid, palmitic acid, capric acid, linoleic acid, and the like.

Many other solvents are possible. One of ordinary skill in the art will find it routine to identify solvents for rapamycin based on the teachings herein.

solubilizer

Generally, any solubilizing agent or combination of solubilizing agents can be used in the liquid formulations described herein.

In some variations, the solubilizing agent is a surfactant or a combination of surfactants. Many surfactants are possible. Combinations of surfactants can also be used, including combinations of various types of surfactants. For example, nonionic, anionic (such as soaps, sulfonates), cationic (such as CTAB), zwitterionic, polymeric or amphoteric surfactants can be used.

Useful surfactants can be identified by mixing the therapeutic agent of interest with a putative solvent and a putative surfactant and observing the properties of the formulation after exposure to the medium.

Examples of surfactants include, but are not limited to, fatty acid esters or amides or ether analogs, or hydrophilic derivatives thereof; monoesters or diesters, or hydrophilic derivatives thereof; or mixtures thereof; monoglycerides or diglycerides, or hydrophilic derivatives thereof; or mixtures thereof; mixtures having an enrichment of monoglycerides or/and diglycerides, or hydrophilic derivatives thereof; surfactants partially derivatized with a hydrophilic moiety; other alcohols, polyols, sugars or oligo- or polysaccharides, monoesters or diesters or polyesters, alkylene oxide oligomers or polymers or block polymers thereof, or hydrophilic derivatives thereof, or amide analogs thereof; fatty acid derivatives of amines, polyamines, polyimines, amino alcohols, amino sugars, hydroxyalkylamines, hydroxypolyamines, peptides, polypeptides or ether analogs thereof.

The hydrophilic-lipophilic balance ("HLB") expresses the relative simultaneous attraction of a surfactant to water and oil (or to both phases of the emulsion system).

Surfactants are characterized by the balance between the hydrophilic and lipophilic parts of their molecules. The hydrophilic-lipophilic balance (HLB) number indicates the polarity of a molecule on a scale of 1-40, with the most commonly used emulsifiers having values between 1-20. HLB increases with increasing hydrophilicity.

Surfactants that can be used include, but are not limited to, surfactants having an HLB greater than 10, 11, 12, 13, or 14. Examples of surfactants include polyoxyethylene products of hydrogenated vegetable oils, polyethoxylated castor oil or polyethoxylated hydrogenated castor oil, polyoxyethylene-sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, and the like, such as Nikkol HCO-50, Nikkol HCO-35, Nikkol HCO-40, Nikkol HCO-60 (from Nikko Chemicals Co. Ltd.); Cremophor (from BASF) such as Cremophor RH40, Cremophor RH60, Cremophor EL, TWEENs (from ICI Chemicals), such as Tween 20, Tween 21, Tween 40, Tween 60, Tween 80, Tween 81, Cremophor RH410, Cremophor RH 455, and the like.

The surfactant component can be selected from compounds having at least one ether formed from at least about 1 to 100 ethylene oxide units and at least one fatty alcohol chain having at least about 12 to about 22 carbon atoms; compounds having at least one ester formed from at least about 1 to 100 ethylene oxide units and at least one fatty acid chain having at least about 12 to 22 carbon atoms; compounds having at least one ether, ester or amide formed from at least about 1 to 100 ethylene oxide units and at least one vitamin or vitamin derivative; and combinations thereof consisting of not more than two surfactants.

Other examples of surfactants include Lumulse GRH-40, TGPS, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polyoxyethylene (20) sorbitan monooleate, glyceryl glycol esters, polyethylene glycol esters, polyglycolyzed glycerides, and the like or mixtures thereof; polyethylene sorbitan fatty acid esters, polyoxyethylene glycerides such as Tagat TO, Tagat L, Tagat I, Tagat I2, and Tagat O (commercially available from Goldschmidt Chemical Co., Essen, Germany); ethylene glycol esters, such as ethylene glycol stearate and ethylene glycol distearate; propylene glycol esters, such as propylene glycol myristate; fatty acid glycerides, such as glyceryl stearate and glyceryl monostearate; sorbitan esters, such as Spans and Tweens; polyglycerol esters, such as polyglyceryl 4-oleate; fatty alcohol ethoxylates, such as Brij-type emulsifiers; ethoxylated propoxylated block copolymers, such as poloxamers; polyethylene glycol esters of fatty acids, such as PEG 300 linoleic acid glyceride or Labrafil 2125CS, PEG 300 oleic acid glyceride or Labrafil M1944CS, PEG 400 caprylic/capric glyceride or Labrasol and PEG 300 caprylic/capric glyceride or Softigen 767; cremophors, such as Cremophor E, Polyoxyl 35 Castor Oil or Cremophor EL, Cremophor EL-P, Cremophor RH4OP, polyoxyl 40 hydrogenated castor oil, Cremophor RH40; polyoxyl 60 hydrogenated castor oil or Cremophor RH60, monocaprylic/capric glyceride, such as Campmul CM10; polyoxyethylated fatty acids (PEG-stearate, PEG-laurate), polyoxyethylated fatty acid glycerides, polyoxyethylated glycerol fatty acid esters, such as Solutol HS-15; PEG-ether sorbitan derivatives (Tween), sorbitan monooleate or Span 20, aromatic compound PEG-glyceride (PECEOL ^™ ), PEG-PPG (polypropylene glycol) copolymer (PLURONICS, including but not limited to PLURONICS F108, F127 and F68, poloxamers, Jeffamines), Tetronics, polyglycerol, PEG-tocopherol, PEG-LICOL 6-oleate; propylene glycol derivatives, alkyl and acyl derivatives of sugars and polysaccharides (octyl sucrose, sucrose stearate, lauroyl dextran, etc.) and/or mixtures thereof; surfactants based on oleic acid or laurate copolymerized with polyols and ethylene oxide; Labrasol Gelucire 44/14; polyoxyethylene stearate; saturated polyglycolyzed glycerides; or poloxamers; all of which are commercially available. Polyoxyethylene sorbitan fatty acid esters may include polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Polyoxyethylene stearates may include polyoxyethylene 6 stearate, polyoxyethylene 8 stearate, polyoxyethylene 12 stearate, and polyoxyethylene 20 stearate. Saturated polyglycolyzed glycerides are, for example, GELUCIRE 44/14 or GELUCIRE ^™ 50/13 (Gattefosse, Westwood, NJ, USA).Poloxamers used herein include poloxamer 124 and poloxamer 188.

Surfactants include d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), polyoxyethylene 8 stearate (PEG400 monostearate), polyoxyethylene 40 stearate (PEG1750 monostearate), and peppermint oil.

In some variations, surfactants with an HLB lower than 10 are used. Such surfactants may optionally be used in combination with other surfactants used as co-surfactants. Some surfactants, mixtures, and other equivalent compositions with an HLB less than or equal to 10 are propylene glycol, glyceryl fatty acids, glyceryl fatty acid esters, polyethylene glycol esters, glyceryl glycol esters, polyglycolized glycerides, and polyoxyethylene steryl ethers. Propylene glycol esters or partial esters form components of commercial products such as Lauroglycol FCC (which contains propylene glycol laurate). The commercially available excipient Maisine 35-1 contains long-chain fatty acids, such as linoleic acid glyceride. Products containing polyoxyethylene stearate ethers, such as Acconon E, may also be used. Labrafil M1944CS is an example of a surfactant in which the composition contains a mixture of glyceryl glycol esters and polyethylene glycol esters.

Rapamycin solubilizer

A variety of solubilizing agents can be used with rapamycin, including but not limited to those described above under "Solubilizing Agents."

In some variations, the solubilizing agent is a surfactant. Non-limiting examples of surfactants that can be used with rapamycin include, but are not limited to, surfactants having an HLB greater than 10, 11, 12, 13, or 14. A non-limiting example is Cremophor EL. In some variations, the surfactant can be a polymeric surfactant, including, but not limited to, PLURONICS F108, F127, and F68 and Tetronics. Some of the solvents used herein can also serve as surfactants. One of ordinary skill in the art will find it routine to identify which solubilizing agents and surfactants can be used with rapamycin based on the teachings herein.

Viscosity modifiers

The liquid formulations described herein may be administered with or further contain a viscosity modifier.

An exemplary viscosity modifier that can be used is hyaluronic acid. Hyaluronic acid is a glycosaminoglycan. It is composed of repeating sequences of glucuronic acid and glucosamine. Hyaluronic acid is present in many tissues and organs of the body and contributes to the viscosity and consistency of such tissues and organs. Hyaluronic acid is present in the eye (including the vitreous body of the eye) and contributes to its viscosity together with collagen. The liquid formulations described herein may also contain hyaluronic acid or be administered together with it.

Other non-limiting examples of viscosity modifiers include polyalkylene oxides, glycerol, carboxymethylcellulose, sodium alginate, chitosan, dextran, dextran sulfate, and collagen.These viscosity modifiers may be chemically modified.

Other viscosity modifiers that can be used include, but are not limited to, carrageenan, cellulose gel, colloidal silica, gelatin, propylene carbonate, carbonic acid, alginic acid, agar, carboxyvinyl polymer or carbomer and polyacrylamide, gum arabic, ester gum, guar gum, gum arabic, ghatti, karaya gum, tragacanth gum, terra, pectin, tamarind, larch arabogalactan, alginates, locust bean, xanthan gum, starch, magnesium aluminum silicate, tragacanth gum, polyvinyl alcohol, gellan gum, hydrocolloid blends, and povidone. Other viscosity modifiers known in the art can also be used, including, but are not limited to, sodium carboxymethylcellulose, sodium alginate, carrageenan, galactomannan, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyethylene glycol, polyvinyl pyrrolidone, sodium carboxymethyl chitosan, sodium carboxymethyl dextran, sodium carboxymethyl starch, xanthan gum, and zein.

Other ingredients in liquid preparations

The formulations described herein may also include a variety of other ingredients, such as stabilizers. Stabilizers that can be used in the formulations described herein include, but are not limited to, agents that (1) improve the compatibility of the excipient with the encapsulating material, such as gelatin, (2) improve the stability of the therapeutic agent, such as rapamycin and/or rapamycin derivative (e.g., prevent crystal growth of the therapeutic agent, such as rapamycin), and/or (3) improve the stability of the formulation. Note that there is overlap between ingredients that are stabilizers and ingredients that are solvents, solubilizers, or surfactants, and that the same ingredient can serve more than one role.

Stabilizers can be selected from fatty acids, fatty alcohols, alcohols, long-chain fatty acid esters, long-chain ethers, hydrophilic derivatives of fatty acids, polyvinyl pyrrolidone, polyvinyl ether, polyvinyl alcohol, hydrocarbons, hydrophobic polymers, hygroscopic polymers and combinations thereof. Amide analogs of the above-mentioned stabilizers can also be used. The selected stabilizer can change the hydrophobicity of the formulation (e.g., oleic acid, wax) or promote the mixing of multiple ingredients in the formulation (e.g., ethanol), control the moisture level of the formulation (e.g., PVP), control the mobility of the phase (substances with a melting point higher than room temperature, such as long-chain fatty acids, alcohols, esters, ethers, amides, etc. or mixtures thereof; wax) and/or promote the compatibility of the formulation with the encapsulating material (e.g., oleic acid or wax). Some of these stabilizers can be used as solvents/cosolvents (e.g., ethanol). The stabilizer can be present in sufficient amounts to inhibit the crystallization of the therapeutic agent (e.g., rapamycin).

Examples of stabilizers include, but are not limited to, saturated, monoolefinic, polyolefinic, branched, cyclic, alkyne-containing, dicarboxylic and functional group-containing fatty acids such as oleic acid, caprylic acid, capric acid, caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), DHA; fatty alcohols such as stearyl alcohol, cetyl alcohol, ceteryl alcohol; other alcohols such as ethanol, isopropanol, butanol; long-chain fatty acid esters, ethers or amides such as glyceryl stearate, cetyl stearate, oleyl ether, stearyl ether, cetyl ether, oleyl amide, stearyl amide; hydrophilic derivatives of fatty acids such as polyglyceryl fatty acids, polyethylene glycol fatty acid esters; polyvinyl pyrrolidone, polyvinyl alcohol (PVA), waxes, docosahexaenoic acid and dehydroabietic acid, etc.

The formulation may also contain a gelling agent, which changes the texture of the final formulation by forming a gel.

As described herein, therapeutic agents (such as rapamycin) can be subjected to conventional pharmaceutical operations (such as sterilization), and compositions containing therapeutic agents may also contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Therapeutic agents can also be formulated with pharmaceutically acceptable excipients for clinical use for the manufacture of pharmaceutical compositions. Preparations for eye administration can exist as solutions, suspensions, solid material particles, separated blocks of solid material, incorporated into polymer matrices, liquid preparations or any other forms of eye administration. Therapeutic agents can be used to prepare medicines for treating, preventing, suppressing, delaying the occurrence of any disease described herein or causing it to disappear. In some variations, therapeutic agents can be used to prepare medicines for treating any disease described herein.

Compositions containing therapeutic agents (such as rapamycin) may contain one or more adjuvants suitable for the specified route of administration. Adjuvants with which the therapeutic agent may be mixed include, but are not limited to, lactose, glucose, starch powder, cellulose alkanoate, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gum arabic, gelatin, sodium alginate, polyvinyl pyrrolidine, and/or polyvinyl alcohol. When a solubilized formulation is desired, the therapeutic agent may be in a solvent including, but not limited to, polyethylene glycol of varying molecular weights, propylene glycol, colloidal solutions of carboxymethyl cellulose, methanol, ethanol, DMSO, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or a variety of buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art and can be used in the practice of the methods, compositions, and liquid formulations described herein. The carrier or diluent may include a time-delaying material (such as glyceryl monostearate or glyceryl distearate, alone or with a wax) or other materials known in the art. Formulations for the uses described herein may also include gel formulations, erodible and non-erodible polymers, microspheres, and liposomes.

Other adjuvants and excipients that may be used include, but are not limited to, _C8 - _C10 fatty acid esters such as softigen 767, polysorbate 80, PLURONICS, Tetronics, Miglyol, and Transcutol.

Commonly used additives and diluents in the pharmaceutical field can be optionally added to pharmaceutical compositions and liquid preparations. These additives and diluents include thickeners, granulating agents, dispersants, flavoring agents, sweeteners, colorants and stabilizers (including pH stabilizers), other excipients, antioxidants (e.g., tocopherol, BHA, BHT, TBHQ, tocopheryl acetate, ascorbyl palmitate, propyl ascorbyl gallate, etc.), preservatives (e.g., parabens), etc. Exemplary preservatives include, but are not limited to, benzyl alcohol, ethanol, benzalkonium chloride, phenol, chlorobutanol, etc. Some useful antioxidants provide oxygen or peroxide inhibitors for the preparation and include, but are not limited to, butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, ascorbyl palmitate, α-tocopherol, etc. Thickeners such as lecithin, hydroxypropyl cellulose, aluminum stearate, etc. can improve the texture of the preparation.

In some variations, the therapeutic agent is rapamycin, and the rapamycin is formulated as a solid or liquid rapamycin. In some variations, the rapamycin is formulated for oral administration.

Additionally, a viscous polymer may be added to the suspension to aid positioning and facilitate placement and handling. In some applications of liquid formulations, a pocket may be surgically formed in the sclera to receive the injection of the liquid formulation. The hydrogel structure of the sclera may act as a rate-controlling membrane. The therapeutic agent particles used to form the suspension may be manufactured by known methods, including, but not limited to, ball milling using ceramic beads. For example, a Cole Parmer ball mill such as the Labmill 8000 may be used with 0.8 mm YTZ ceramic beads from Tosoh or Norstone Inc.

The formulations are conveniently presented in unit dosage form and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of combining the therapeutic agent with a pharmaceutical carrier or excipient. The formulations can be prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if desired, shaping the product.

In some variations, the formulations described herein are provided in one or more unit dosage forms, wherein the unit dosage forms comprise an amount of a liquid formulation described herein that is effective to treat or prevent the disease or condition for which it is administered. In some variations, the formulations described herein are provided in one or more unit dosage forms, wherein the unit dosage forms comprise an amount of a liquid rapamycin formulation described herein that is effective to treat or prevent the disease or condition for which it is administered.

In some embodiments, the unit dosage form is formulated at the concentration at which it will be administered. In some variations, the unit dosage form is diluted prior to administration to a subject. In some variations, the liquid formulations described herein are diluted in an aqueous medium prior to administration to a subject. In some variations, the aqueous medium is an isotonic medium. In some variations, the liquid formulations described herein are diluted in a non-aqueous medium prior to administration to a subject.

Another aspect of the present invention provides a medicine box comprising one or more unit dosage forms described herein. In some embodiments, the medicine box comprises one or more packages and instructions for treating one or more diseases or conditions. In some embodiments, the medicine box comprises a diluent that is not physically in contact with the formulation or pharmaceutical preparation. In some embodiments, the medicine box comprises any one or more unit dosage forms described herein in one or more sealed tubes. In some embodiments, the medicine box comprises any one or more sterile unit dosage forms.

In some variations, the unit dosage form is a container, including but not limited to a sterile sealed container. In some variations, the container is a vial, ampoule or small volume applicator, including but not limited to a syringe. In some variations, the small volume applicator is pre-filled with rapamycin for treating ophthalmic diseases or conditions, including but not limited to limus compounds for treating age-related macular degeneration. Described herein are pre-filled small volume applicators that are pre-filled with a preparation containing a therapeutic agent (including but not limited to rapamycin). In some variations, the small volume applicator is pre-filled with a solution containing a therapeutic agent (including but not limited to rapamycin) and polyethylene glycol, and optionally further comprises one or more additional ingredients, including but not limited to ethanol. In some variations, the pre-filled small volume applicator is pre-filled with a solution containing about 2% rapamycin, about 94% PEG-400, and about 4% ethanol.

Described herein are medicine boxes comprising one or more containers. In some variations, the medicine box comprising one or more small volume applicators is pre-filled with a formulation containing a therapeutic agent as described herein, including but not limited to a formulation comprising rapamycin, a formulation comprising rapamycin and polyethylene glycol and optionally further comprising one or more additional ingredients (the additional ingredients include but are not limited to ethanol), and a formulation in liquid form comprising about 2% rapamycin, about 94% PEG-400, and about 4% ethanol. In some variations, the medicine box comprises one or more containers (including but not limited to pre-filled small volume applicators) and instructions for use thereof. In another variation, the medicine box comprises one or more small volume applicators pre-filled with rapamycin and instructions for use thereof for treating eye diseases or conditions. In some variations, the container described herein is in secondary packaging.

Route of administration

The compositions, methods, and liquid formulations described herein deliver one or more therapeutic agents to a subject (including but not limited to a human subject).

In some variations, the compositions, methods, and liquid formulations described herein deliver one or more therapeutic agents to a human subject in an aqueous medium.

In some variations, the compositions, methods, and liquid formulations described herein deliver one or more therapeutic agents to an aqueous medium in or near the area of the disease or condition to be treated, prevented, inhibited, delayed in onset, or caused to regress.

In some variations, the compositions, methods, and liquid formulations described herein deliver one or more therapeutic agents to the eye of a subject, including the macula and retinal choroidal tissue, in an amount and for a duration effective to treat, prevent, inhibit, delay the onset of, or cause regression of the diseases and conditions described in the "Diseases and Conditions" section.

[0014] "Retina choroid" and "retinal choroidal tissue" are used synonymously herein and refer to the combined retinal and choroidal tissue of the eye.

As non-limiting examples, compositions described herein, liquid preparation and method can be applied to following tissue directly or by periocular approach: vitreous body, aqueous humor, sclera, conjunctiva, between sclera and conjunctiva, retinal choroid tissue, macula or in experimenter's eye or other areas near eye with the amount and the persistent period that effectively treats, prevents, delays CNV and wet AMD generation or makes it disappear, and can be different for each different delivery site.

Intravitreal administration is more invasive than some other types of ocular procedures. Because of the risk of possible adverse effects, intravitreal administration is not optimal for treating relatively healthy eyes. In contrast, periocular administration (such as subconjunctival administration) is much less invasive than intravitreal administration. When the therapeutic agent is delivered via the periocular route, patients with relatively healthy eyes can be treated instead of those who can be treated using intravitreal administration. In some variations, subconjunctival injection is used to prevent or delay the onset of a disease or condition of the eye, where the subject's eye has a visual acuity of 20/40 or better.

As used herein, "subconjunctival" placement or injection refers to placement or injection between the sclera and the conjunctiva. Subconjunctival is sometimes referred to herein as "sub-conj" administration.

Routes of administration that can be used to administer the liquid formulation include, but are not limited to, placing the liquid formulation in an aqueous medium in a subject (e.g., by injection), including, but not limited to, placing the liquid formulation in the eye of a subject (including, but not limited to, a human subject) (including, but not limited to, by injection). The liquid formulation can be administered systemically, including, but not limited to, the following delivery routes: rectal, vaginal, instillation, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, intracisternal, epidermal, subcutaneous, intradermal, transdermal, intravenous, intrajugular, intraabdominal, intracranial, intraocular, intrapulmonary, intrathoracic, intratracheal, nasal, buccal, sublingual, oral, parenteral, or sprayed or atomized using an aerosol spray.

Compositions and liquid formulations containing therapeutic agents can be administered directly to the eye using a variety of procedures, including, but not limited to, procedures in which (1) the therapeutic agent is administered by injection using a syringe and hypodermic needle, (2) the therapeutic agent is injected using a specially designed device, and (3) a pocket is surgically created within the sclera to serve as a reservoir for the therapeutic agent or therapeutic agent composition prior to injection of the therapeutic agent. For example, in one administration procedure, a surgeon creates a pocket within the sclera of the eye and then injects a solution or liquid formulation containing the therapeutic agent into the pocket.

Other administration procedures include, but are not limited to, procedures in which (1) the therapeutic agent formulation is injected through a specially designed curved cannula to place the therapeutic agent directly against a portion of the eye, (2) a compressed form of the therapeutic agent is placed directly against a portion of the eye, (3) the therapeutic agent is inserted into the sclera via a specially designed syringe or inserter, (4) a liquid formulation containing the therapeutic agent is incorporated into a polymer, (5) the surgeon creates a small conjunctival incision through which sutures and any therapeutic agent delivery structure are passed to secure the structure adjacent to the sclera, and (6) a needle is used for injection directly into the vitreous of the eye or into any other site described.

The liquid formulations described herein can be used directly (eg, by injection), as an elixir for topical administration (including but not limited to eye drops), or in soft or hard gelatin or starch capsules. The capsules can be banded to prevent leakage.

Delivered by injection

One method for delivering the compositions and liquid formulations described herein is by injection. In this method, the compositions and liquid formulations can be injected into a subject (including but not limited to a human subject), or injected into a location in or near the subject's eye for delivery to the subject or subject's eye. Injection includes but is not limited to intraocular and periocular injections. Non-limiting examples of locations in or near the subject's eye are as follows.

Injection of therapeutic agents into the vitreous can provide high local concentrations of the therapeutic agent in the vitreous and retina. In addition, it has been found that the elimination half-life of drugs in the vitreous increases with molecular weight.

Intracameral injection or injection into the anterior chamber may also be used. In one example, up to 100 μl may be injected intracamerally.

Periocular routes of delivery can deliver therapeutic agents to the retina without some of the risks of intravitreal delivery. Periocular routes include, but are not limited to, subconjunctival, subtenon, retrobulbar, periocular, and postjuxtascleral delivery. A "periocular" delivery route refers to placement near or around the eye. For an exemplary description of periocular routes for retinal drug delivery, see Periocular routes for retinal drug delivery, Raghava et al. (2004), Expert Opin. Drug Deliv. 1(1):99-114, which is incorporated herein by reference in its entirety.

In some variations, the liquid formulations described herein are administered intraocularly. Intraocular administration includes placement or injection into the eye (including but not limited to the vitreous).

Subconjunctival injection can be performed by injecting the therapeutic agent into the conjunctiva, or between the sclera and the conjunctiva. In one embodiment, up to about 500 μl can be injected subconjunctival. As a non-limiting example, a needle of about 25 to 30 gauge and about 30 mm long can be used. The local pressure at the subconjunctival site where the therapeutic agent is administered can improve the delivery of the therapeutic agent to the posterior segment by reducing local choroidal blood flow.

Subtenon injections can be administered by injecting the therapeutic agent into the tenon's capsule surrounding the upper portion of the eye and into the "belly" of the superior rectus muscle. In one example, up to about 4 ml can be injected subtenonally. As a non-limiting example, a blunt-tipped cannula about 2.5 cm long can be used.

Retrobulbar injection refers to the injection into the conical compartment of the four rectus muscles and their myocardial septa at the back of the eyeball. In one embodiment, up to about 5 ml can be injected retrobulbarly. As a non-limiting example, a blunt needle of about 25 or about 27 gauge can be used.

Peribular injections can be made into locations outside the four rectus muscles and their septal regions (i.e., outside the cone of the muscle). Volumes of up to about 10 ml can be injected peribulbarly. As a non-limiting example, a blunt-tipped cannula about 1.25 inches long and about 25 gauge can be used.

Post-juxtascleral delivery refers to placing the therapeutic agent near or on the macula, in direct contact with the outer surface of the sclera, and without puncturing the eyeball. In one example, up to about 500 ml can be injected post-juxtascleral. As a non-limiting example, a blunt-tipped curved cannula (particularly one designed with a 56° angle) is used to place the therapeutic agent in the scleral incision.

In some variations, the liquid formulations described herein are injected intraocularly. Intraocular injection includes injection into the eye.

Sites to which the compositions and liquid formulations can be applied include, but are not limited to, the vitreous body, aqueous humor, sclera, conjunctiva, between the sclera and conjunctiva, retinal choroidal tissue, macula, or other areas in or near the subject's eye. Methods for placing the compositions and liquid formulations include, but are not limited to, injection.

In one method that can be used, the therapeutic agent is dissolved in a solvent or solvent mixture and then injected into or near the vitreous, aqueous humor, sclera, conjunctiva, between the sclera and conjunctiva, retinal choroidal tissue, macula, or other area in or near the eye, or other medium in the subject according to any of the procedures described above. In one such method that can be used, the therapeutic agent is rapamycin in a liquid formulation.

When the therapeutic agent is rapamycin, compositions and liquid preparations can be used to deliver or maintain a certain amount of rapamycin in ocular tissue, and the ocular tissue includes but is not limited to retina, choroid or vitreous body, and the amount can effectively treat AMD. In a non-limiting example, it is believed that a liquid preparation that delivers a certain amount of rapamycin can be used to treat wet AMD, and the amount can provide a rapamycin concentration of about 0.1pg/ml to about 2μg/ml in the vitreous body. In some non-limiting examples, it is believed that a liquid preparation that delivers about 0.1pg/mg to about 1μg/mg rapamycin concentration in the retinal choroidal tissue can be used to treat wet AMD. Other effective concentrations can be easily determined by those skilled in the art based on teachings described herein.

Method for preparing liquid preparation

A non-limiting method that can be used to prepare the liquid formulations described herein (including but not limited to liquid formulations containing rapamycin) is by mixing the solvent and the therapeutic agent together at room temperature or slightly elevated temperature (optionally using a sonicator) until a solution or suspension is obtained, and then cooling the formulation. Other ingredients (including but not limited to those listed above) can then be mixed with the formulation. Other preparation methods that can be used are described herein, including in the Examples, and those skilled in the art will be able to select other preparation methods based on the teachings herein.

Extended delivery of therapeutic agents

Described herein are compositions and liquid formulations that exhibit in vivo delivery or clearance profiles having one or more of the following characteristics. The delivery or clearance profile is the clearance profile of the therapeutic agent following subconjunctival injection or injection into the vitreous of a rabbit eye. In some variations, the delivery or clearance profile is the clearance profile of rapamycin following subconjunctival injection or injection into the vitreous of a rabbit eye. The volume of the rabbit vitreous is approximately 30-40% of the volume of the human vitreous. The amount of therapeutic agent is measured using techniques described in Example 2, but is not limited by the formulations and therapeutic agents described in Example 2.

In some variations, therapeutic agents having in vivo delivery or clearance profiles as described herein include, but are not limited to, those described in the "Therapeutic Agents" section. In some variations, the therapeutic agent is rapamycin. In some variations, the liquid formulations described herein are used to deliver the therapeutic agent at a concentration equivalent to rapamycin. The liquid formulations described herein may contain any therapeutic agent, including, but not limited to, the therapeutic agents described in the "Therapeutic Agents" section, at a concentration equivalent to rapamycin (including, but not limited to, the concentrations described herein, including in the Examples).

The "mean in vivo percent" level refers to the average concentration of the therapeutic agent obtained from multiple rabbit eyes for a given time point, and is obtained by dividing the mean concentration of the therapeutic agent at one time point by the mean concentration of the therapeutic agent at another time point. In some variations of the mean in vivo percent level, the therapeutic agent is rapamycin.

The average concentration of the therapeutic agent in rabbit ocular tissue at a given time after administration of a formulation containing the therapeutic agent can be measured according to the following method: When injecting a volume of less than 10 μl, a Hamilton syringe is used.

The liquid preparation should be stored at 2-8°C before use.

The experimental animals were specific pathogen-free (SPF) New Zealand White rabbits. A mixed population of approximately 50% male and 50% female was used. Rabbits were at least 12 weeks of age, typically at least 14 weeks of age, at the time of dosing. Each rabbit weighed at least 2.2 kg, typically at least 2.5 kg, at the time of dosing. Animals were quarantined for at least one week prior to the study and general health parameters were examined. No unhealthy animals were used in the study. At a given time point, at least six eyes were measured and averaged.

Housing and sanitation were performed according to standard procedures used in the industry. Animals were provided approximately 150 grams of Teklad Certified Hi-Fiber Rabbit Diet daily and had access to tap water ad libitum. The water was known to be free of contaminants, and no additional analysis was performed beyond that provided by the local water community. Environmental conditions were monitored.

Each animal underwent a pre-treatment ophthalmic examination (slit lamp and ophthalmoscopy) performed by a board-certified veterinary ophthalmologist. Ocular examinations were scored according to the McDonald and Shadduck scoring system described in Dermatoxicology, F.N. Marzulli and H.I. Maibach, 1977 "Eye Irritation," T.O. McDonald and J.A. Shadduck (pp. 579-582). Observations were recorded using a standardized data collection form. Acceptance criteria for the study were as follows: conjunctival injection and swelling scores ≤ 1; all other observed variables scored 0.

Gentamicin eye drops were applied twice daily to both eyes of each animal on the day before, on the day of administration (Day 1), and on the day after administration (Day 2). Administration was carried out in two phases, with the first phase including one group of animals and the second phase including the remaining animals. Animals were randomly divided into masked treatment groups according to a modified Latin square before each administration phase. Animals were fasted for at least 8 hours before injection. The start time of fasting and the injection time were recorded.

Animals were weighed and anesthetized with an intravenous injection of a ketamine/xylazine mixture (87 mg/mL ketamine, 13 mg/mL xylazine) at a volume of 0.1-0.2 mL/kg. Both eyes of each animal were prepared for injection as follows: approximately 5 minutes before injection, the eyes were moistened with ophthalmic betadine. Five minutes later, the betadine was washed out of the eyes with sterile saline. 0.5% proparacaine hydrochloride (1-2 drops) was delivered to each eye. For eyes to be injected intravitreally, 1% tropicamide (1 drop) was delivered to each eye.

On day 1, each animal received an injection of the test article or control into both eyes. Animals in selected groups received a second dose on day 90 ± 1. Dosing was performed subconjunctival or intravitreally. The actual treatment, injection site, and dose volume were masked and revealed at the end of the study.

Subconjunctival injections were performed using an insulin syringe and a 30 gauge x 1/2" needle. Forceps were used to elevate the bulbar conjunctiva in the dorsotemporal quadrant. The test article was injected into the subconjunctival space.

Intravitreal injections were performed using an insulin syringe and a 30-gauge x 1/2-inch needle. For each injection, the needle was introduced through the ventro-nasal quadrant of the eye, 2-3 mm posterior to the limbus, with the bevel of the needle pointing downward and posterior to avoid the lens. The test article was injected as a single bolus into the vitreous near the retina.

Animals were observed twice daily for mortality/morbidity. Animals identified as moribund were euthanized using an intravenous injection of a commercial euthanasia solution. Both eyes were removed and stored frozen at -70°C for possible future evaluation. If an animal died before postmortem rigor mortis developed, both eyes were removed and stored frozen at -70°C for possible future evaluation. Animals found dead after postmortem rigor mortis developed were not necropsied.

Animals were weighed randomly before dosing on day 1 and before euthanasia.

All animals were ophthalmologically observed (slit lamp and indirect ophthalmoscopy) on days 5 ± 1, 30 ± 1, 60 ± 1, and 90 ± 1 (later dates in some variants). Observations were performed by a board-certified veterinary ophthalmologist. For animals to be dosed on day 90 ± 1, ophthalmological observations were performed prior to dosing. Ophthalmological examinations were scored according to the McDonald and Shadduck scoring system described in Dermatoxicology, F.N. Marzulli and H.I. Maibach, 1977 "Eye Irritation," T.O. McDonald and J.A. Shadduck (pp. 579-582), and observations were recorded using a standardized data collection form.

Before necropsy, whole blood samples (1-3 mL per sample) were collected from each animal in EDTA-containing vacutainer tubes. Each tube was filled at least 2/3 and mixed thoroughly for at least 30 seconds. The tubes were kept frozen until shipped on dry ice.

Euthanize the animals using an intravenous injection of a commercially available euthanasia solution. Perform euthanasia according to standard procedures used in the industry.

For the treatment groups that received intravitreal or subconjunctival placebo, all eyes from each of these groups were placed in Davidson's solution for approximately 24 hours. After 24 hours, the eyes were transferred to 70% ethanol; these eyes underwent masked pathological evaluation by a board-certified veterinary pathologist. The time the eyes were placed in Davidson's solution and the time they were removed were recorded.

For groups treated with the test article intravitreally or subconjunctivally, a few eyes from each group were frozen at -70°C and subjected to pharmacokinetic analysis. The remaining eyes from each group were placed in Davidson's solution for approximately 24 hours. After 24 hours, the eyes were transferred to 70% ethanol; these eyes underwent masked histopathological evaluation by a board-certified veterinary pathologist. The time the eyes were placed in Davidson's solution and the time they were removed were recorded.

Frozen samples for pharmacokinetic analysis were dissected using disposable tools. One set of tools was used for each eye and then discarded. The samples were thawed at room temperature for 1 to 2 minutes to ensure that the frost around the tissue was removed. The sclera was dissected into 4 quadrants and the vitreous was removed. If non-dispersed masses (NDM) were clearly visible in the vitreous, the vitreous was separated into two slices. The slice with NDM was approximately two-thirds of the vitreous. The slice without NDM was the part of the vitreous farthest from the NDM. The aqueous humor, lens, iris, and cornea were separated. Retinal choroidal tissue was removed using forceps and collected for analysis. The conjunctiva was separated from the sclera.

The various tissue types were collected into pre-weighed separate vials, which were then capped and weighed. Tissue vials were stored at -80°C until analysis.

Sirolimus levels in the retina, choroid, sclera, vitreous humor, and anticoagulated whole blood were determined by high-pressure liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) using 32-O-desmethoxyrapamycin as an internal standard. When NDM was observed in the vitreous, vitreous sections containing NDM and those without NDM were analyzed separately.

The average therapeutic agent concentration for a period of time is the average concentration at each time point for a representative time point of the period of time. For example, if the time period is 30 days, the average concentration can be measured at 5-day intervals: for the average concentration on day 5, the average of the multiple concentration measurements on day 5 should be calculated; for the average concentration on day 10, the average of the multiple concentration measurements on day 10 should be calculated, and so on.

In some variations, the liquid formulations described herein can have an in vivo delivery profile to the vitreous having the characteristics described below, wherein the delivery profile is used to deliver a therapeutic agent in vivo after the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye. One non-limiting variation of an in vivo delivery profile to the vitreous is shown in FIG2 .

At 40 days after injection, the average percent vitreous level in vivo relative to the level at 20 days after injection can be between about 70% and about 100%, and more often between about 80% and about 90%. At 40 days after injection, the average percent vitreous level in vivo relative to the level at 20 days after injection can be greater than about 70%, and more often greater than about 80%.

At 67 days post-injection, the average percent vitreous level in vivo relative to the level at 20 days post-injection may be between about 75% and about 115%, and more often between about 85% and about 105%. At 67 days post-injection, the average percent vitreous level in vivo relative to the level at 20 days post-injection may be greater than about 75%, and more often greater than about 85%.

At 90 days after injection, the average percent vitreous level in vivo relative to the level at 20 days after injection can be between about 20% and about 50%, and more often between about 30% and about 40%. At 90 days after injection, the average percent vitreous level in vivo relative to the level at 20 days after injection can be greater than about 20%, and more often greater than about 30%.

In some variations, the in vivo mean percent vitreous level relative to the level at day 20 post-injection is characterized by less than about 100% at day 40 post-injection; less than about 115% at day 67 post-injection; and less than about 50% at day 90 post-injection.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of the rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 0.01 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of the rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 0.1 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of the rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 1 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye.

In some variations, the liquid formulations described herein may have an in vivo delivery profile to the retina and choroid characterized by delivering the therapeutic agent in vivo following injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye.

At 40 days after injection, the in vivo mean percentage retinal choroidal level can be between about 350% and about 410%, and more often between about 360% and about 400%, relative to the level expressed at day 20 after injection. At 40 days after injection, the in vivo mean percentage retinal choroidal level can be greater than about 350%, and more often greater than about 360%, relative to the level expressed at day 20 after injection.

At 67 days post-injection, the in vivo mean percentage retinal choroidal level can be between about 125% and about 165%, and more often between about 135% and about 155%, relative to the level expressed at day 20 post-injection. At 67 days post-injection, the in vivo mean percentage retinal choroidal level can be greater than about 125%, and more often greater than about 135%, relative to the level expressed at day 20 post-injection.

At 90 days after injection, the in vivo mean percentage retinal choroidal level can be between about 10% and about 50%, and more often between about 20% and about 40%, relative to the level expressed at day 20 after injection. At 90 days after injection, the in vivo mean percentage retinal choroidal level can be greater than about 10%, and more often greater than about 20%, relative to the level expressed at day 20 after injection.

In some variations, the in vivo mean percent retinal choroidal level relative to the level at day 20 post-injection is characterized by less than about 410% at day 40 post-injection; less than about 165% at day 67 post-injection; and less than about 50% at day 90 post-injection.

In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of at least about 0.001 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous retinal choroidal tissue of the rabbit eye of at least about 0.01 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye.

In some variations, the level of therapeutic agent present in the retinal choroidal tissue first rises, then peaks and then decreases. The peak may occur, for example, around day 40 after injection.

In some variations, a liquid formulation described herein may have an in vivo scleral clearance profile characterized as follows, wherein the clearance profile is the clearance profile of the therapeutic agent in vivo after injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye. When injected between the sclera and conjunctiva, the scleral level is considered to include the injected liquid formulation.

At 40 days after injection, the in vivo average percent scleral level can be between about 150% and about 230%, and more often between about 170% and about 210%, relative to the level expressed at day 20 after injection. At 40 days after injection, the in vivo average percent scleral level can be greater than about 150%, and more often greater than about 170%, relative to the level expressed at day 20 after injection.

At 67 days post-injection, the in vivo average percent scleral level can be between about 30% and about 70%, and more often between about 40% and about 60%, relative to the level expressed at day 20 post-injection. At 67 days post-injection, the in vivo average percent scleral level can be greater than about 30%, and more often greater than about 40%, relative to the level expressed at day 20 post-injection.

At 90 days post-injection, the in vivo average percent scleral level can be between about 110% and about 160%, and more often between about 125% and about 145%, relative to the level expressed at day 20 post-injection. At 90 days post-injection, the in vivo average percent scleral level can be greater than about 110%, and more often greater than about 125%, relative to the level expressed at day 20 post-injection.

In some variations, the in vivo mean percent scleral level relative to the level at day 20 post-injection is characterized by being less than about 230% at day 40 post-injection; less than about 70% at day 67 post-injection; and less than about 160% at day 90 post-injection.

In some variations, the level of therapeutic agent present in the sclera first rises, then peaks and then decreases. The peak may occur, for example, around day 40 after injection.

In some variations, the liquid formulations described herein may have an in vivo delivery profile to the vitreous characterized as follows, wherein the delivery profile is a delivery profile of the therapeutic agent in vivo following injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye.

At 14 days after injection, the average percent vitreous level in vivo can be between about 1350% and about 1650%, and more often between about 1450% and about 1550%, relative to the level expressed at day 2 after injection. At 14 days after injection, the average percent vitreous level in vivo can be greater than about 1350%, and more often greater than about 1450%, relative to the level expressed at day 2 after injection.

At 35 days after injection, the average percent vitreous level in vivo relative to the level expressed on day 2 after injection can be between about 200% and about 300%, and more often between about 225% and about 275%. At 35 days after injection, the average percent vitreous level in vivo relative to the level expressed on day 2 after injection can be greater than about 200%, and more often greater than about 225%.

At 62 days post-injection, the average percent vitreous level in vivo relative to the level expressed on day 2 post-injection can be between about 100% and about 160%, and more often between about 115% and about 145%. At 62 days post-injection, the average percent vitreous level in vivo relative to the level expressed on day 2 post-injection can be greater than about 100%, and more often greater than about 115%.

At 85 days after injection, the average percent vitreous level in vivo can be between about 5% and about 30%, and more often between about 10% and about 25%, relative to the level expressed on day 2 after injection. At 85 days after injection, the average percent vitreous level in vivo can be greater than about 5%, and more often greater than about 10%, relative to the level expressed on day 2 after injection.

In some variations, the mean percent vitreous level in vivo relative to the level expressed on day 2 post-injection is characterized by being less than about 1600% on day 14 post-injection; less than about 300% on day 35 post-injection; less than about 160% on day 62 post-injection and less than about 30% on day 85 post-injection.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 0.01 ng/mL for at least about 30 days, at least about 60 days, or at least about 85 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 0.1 ng/mL for at least about 30 days, at least about 60 days, or at least about 85 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 1 ng/mL for at least about 30 days, at least about 60 days, or at least about 10 days after the liquid formulation is administered to the rabbit eye.

In some variations, the level of therapeutic agent present in the vitreous initially rises, then peaks and then decreases. The peak may occur, for example, around day 14 after injection.

In some variations, the liquid formulations described herein may have an in vivo delivery profile to the retina and choroid characterized by the following delivery profile of the therapeutic agent in vivo following injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye.

At day 35 post-injection, the in vivo mean percentage retinal choroidal level can be between about 320% and about 400%, and more often between about 340% and about 380%, relative to the level expressed at day 14 post-injection. At day 35 post-injection, the in vivo mean percentage retinal choroidal level can be greater than about 320%, and more often greater than about 340%, relative to the level expressed at day 14 post-injection.

At 62 days post-injection, the in vivo mean percentage retinal choroidal level can be between about 3% and about 25%, and more often between about 6% and about 20%, relative to the level expressed at day 14 post-injection. At 62 days post-injection, the in vivo mean percentage retinal choroidal level can be greater than about 3%, and more often greater than about 6%, relative to the level expressed at day 14 post-injection.

At 85 days post-injection, the in vivo mean percent retinal choroidal level relative to the level expressed at day 14 post-injection may be between about 0.1% and about 6%, and more often between about 0.5% and about 4%. At 85 days post-injection, the in vivo mean percent retinal choroidal level relative to the level expressed at day 14 post-injection may be greater than about 0.1%, and more often greater than about 0.5%.

In some variations, the in vivo mean percent retinal choroidal level relative to the level at day 14 post-injection is characterized by less than about 400% at day 35 post-injection; less than about 25% at day 62 post-injection; and less than about 6% at day 85 post-injection.

In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of at least about 0.001 ng/mL for at least about 30 days, at least about 60 days, or at least about 85 days after administering the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of at least about 0.01 ng/mL for at least about 30 days, at least about 60 days, or at least about 85 days after administering the liquid formulation to the rabbit eye.

In some variations, a liquid formulation described herein may have an in vivo scleral clearance profile characterized as follows, wherein the clearance profile is the clearance profile of the therapeutic agent in vivo after injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye. When injected between the sclera and conjunctiva, the scleral level is considered to include the injected liquid formulation.

At day 35 post-injection, the in vivo average percent scleral level can be between about 0.1% and about 0.7%, and more often between about 0.2% and about 0.6%, relative to the level expressed at day 14 post-injection. At day 35 post-injection, the in vivo average percent scleral level can be greater than about 0.1%, and more often greater than about 0.2%, relative to the level expressed at day 14 post-injection.

At 62 days post-injection, the in vivo average percent scleral level relative to the level expressed at day 14 post-injection may be between about 0.05% and about 0.35%, and more often between about 0.07% and about 0.3%. At 62 days post-injection, the in vivo average percent scleral level relative to the level expressed at day 14 post-injection may be greater than about 0.05%, and more often greater than about 0.07%.

At 85 days post-injection, the in vivo average percent scleral level relative to the level expressed at day 14 post-injection may be between about 0.1% and about 0.9%, and more often between about 0.3% and about 0.7%. At 85 days post-injection, the in vivo average percent scleral level relative to the level expressed at day 14 post-injection may be greater than about 0.1%, and more often greater than about 0.3%.

In some variations, the in vivo mean percent scleral level relative to the level at day 14 post-injection is characterized by less than about 0.7% at day 35 post-injection; less than about 0.35% at day 62 post-injection; and less than about 0.9% at day 85 post-injection.

In some variations, the liquid formulations described herein may have an in vivo vitreous clearance profile characterized as follows, wherein the clearance profile is the in vivo clearance profile of the therapeutic agent following injection of the liquid formulation into the vitreous of a rabbit eye. When injected into the vitreous, the measured vitreous levels are considered to include the injected formulation.

At 35 days after injection, the average percent vitreous level in vivo can be between about 1% and about 40%, and more often between about 1% and about 10%, relative to the level expressed at day 14 after injection. At 35 days after injection, the average percent vitreous level in vivo can be greater than about 1% relative to the level expressed at day 14 after injection.

At 62 days post-injection, the average percent vitreous level in vivo can be between about 1% and about 40%, and more often between about 5% and about 25%, relative to the level expressed at day 14 post-injection. At 62 days post-injection, the average percent vitreous level in vivo can be greater than about 1%, and more often greater than about 5%, relative to the level expressed at day 14 post-injection.

At 90 days after injection, the average percent vitreous level in vivo can be between about 1% and about 40%, and more often between about 10% and about 30%, relative to the level expressed at day 14 after injection. At 90 days after injection, the average percent vitreous level in vivo can be greater than about 1%, and more often greater than about 10%, relative to the level expressed at day 14 after injection.

In some variations, the level of therapeutic agent present in the vitreous initially rises, then peaks and then decreases. The peak may occur, for example, around day 14 after injection.

In some variations, the liquid formulations described herein may have an in vivo delivery profile to the retina and choroid characterized as follows, wherein the delivery profile is a delivery profile of the therapeutic agent in vivo following injection of the liquid formulation into the vitreous of a rabbit eye.

At day 35 post-injection, the in vivo mean percentage retinal choroidal levels can be between about 3400% and about 5100%, and more often between about 3750% and about 4750%, relative to the levels expressed at day 14 post-injection. At day 35 post-injection, the in vivo mean percentage retinal choroidal levels can be greater than about 3400%, and more often greater than about 3750%, relative to the levels expressed at day 14 post-injection.

At 62 days post-injection, the in vivo mean percent retinal choroidal level may be between about 0.1% and about 5%, and more often between about 1% and about 3%, relative to the level expressed at day 14 post-injection. At 62 days post-injection, the in vivo mean percent retinal choroidal level may be greater than about 0.1%, and more often greater than about 1%, relative to the level expressed at day 14 post-injection.

At 90 days after injection, the in vivo mean percentage retinal choroidal level can be between about 10% and about 50%, and more often between about 20% and about 40%, relative to the level expressed at day 14 after injection. At 90 days after injection, the in vivo mean percentage retinal choroidal level can be greater than about 10%, and more often greater than about 20%, relative to the level expressed at day 14 after injection.

In some variations, the in vivo mean percent retinal choroidal level relative to the level at day 14 post-injection is characterized by less than about 5100% at day 35 post-injection; less than about 5% at day 62 post-injection; and less than about 50% at day 90 post-injection.

In some variations, the liquid formulations described herein may have an in vivo delivery profile to the sclera characterized by the following delivery profile of the therapeutic agent in vivo following injection of the liquid formulation into the vitreous of a rabbit eye.

At day 35 post-injection, the in vivo average percent scleral level can be between about 1700% and about 2600%, and more often between about 1900% and about 2400%, relative to the level expressed at day 14 post-injection. At day 35 post-injection, the in vivo average percent scleral level can be greater than about 1700%, and more often greater than about 1900%, relative to the level expressed at day 14 post-injection.

At 62 days post-injection, the in vivo average percent scleral level can be between about 120% and about 180%, and more often between about 140% and about 160%, relative to the level expressed at day 14 post-injection. At 62 days post-injection, the in vivo average percent scleral level can be greater than about 120%, and more often greater than about 140%, relative to the level expressed at day 14 post-injection.

At 90 days post-injection, the in vivo mean percent scleral level relative to the level expressed at day 14 post-injection may be between about 95% and about 155%, and more often between about 115% and about 135%. At 90 days post-injection, the in vivo mean percent scleral level relative to the level expressed at day 14 post-injection may be greater than about 95%, and more often greater than about 115%.

In some variations, the in vivo mean percent scleral level relative to the level at day 14 post-injection is characterized by less than about 2600% at day 35 post-injection; less than about 180% at day 62 post-injection; and less than about 155% at day 90 post-injection.

In some variations, the liquid formulation is injected into the vitreous humor of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of at least about 0.001 ng/mL of the therapeutic agent in the sclera of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected into the vitreous humor of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of at least about 0.01 ng/mL of the therapeutic agent in the sclera of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected into the vitreous humor of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of at least about 0.1 ng/mL of the therapeutic agent in the sclera of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye.

In some variations, the level of therapeutic agent present in the vitreous initially rises, then peaks and then decreases. The peak may occur, for example, around day 35 after injection.

In some variations, the in situ gelling formulations described herein may have an in vivo delivery profile to the vitreous characterized by delivery of the therapeutic agent in vivo following injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye.

At 32 days post-injection, the average percent vitreous level in vivo can be between about 25% and about 85%, and more often between about 45% and about 65%, relative to the level expressed at day 7 post-injection. At 40 days post-injection, the average percent vitreous level in vivo can be greater than about 25%, and more often greater than about 45%, relative to the level expressed at day 7 post-injection.

At 45 days post-injection, the average percent vitreous level in vivo can be between about 2% and about 50%, and more often between about 8% and about 20%, relative to the level expressed at day 7 post-injection. At 67 days post-injection, the average percent vitreous level in vivo can be greater than about 2%, and more often greater than about 5%, relative to the level expressed at day 7 post-injection.

At 90 days after injection, the average percent vitreous level in vivo relative to the level expressed at 7 days after injection can be between about 40% and about 100%, and more often between about 60% and about 80%. At 90 days after injection, the average percent vitreous level in vivo relative to the level expressed at 7 days after injection can be greater than about 40%, and more often greater than about 60%.

In some variations, the mean percent vitreous level in vivo relative to the level at day 7 post-injection is characterized by being less than about 80% at day 32 post-injection; less than about 30% at day 45 post-injection; and less than about 100% at day 90 post-injection.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 0.1 pg/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 0.01 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least about 0.1 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 1 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 10 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least 0.001 ng/mL in the vitreous of the rabbit eye for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least 0.01 ng/mL in the vitreous of the rabbit eye for at least about 30 days, at least about 60 days, at least about 90 days, or at least about 120 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent of at least 0.1 ng/mL in the vitreous of the rabbit eye for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least 0.5 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 0.001 ng/mL and 10.0 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 0.01 ng/mL and 10.0 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 0.1 ng/mL and 10 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 0.5 ng/mL and 10.0 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye of less than 100 within 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye of less than 50 within 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye of less than 10 within 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye such that a ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye is less than 5 within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

As used herein, "substantially constant" means that the average level does not change by more than an order of magnitude over an extended period of time, ie the difference between the maximum and minimum average concentrations measured at different times during the relevant period of time is less than a factor of 10.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye that remains approximately constant at a value greater than 0.001 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the solution is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye that remains approximately constant at a value greater than 0.01 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the vitreous of the rabbit eye that remains approximately constant at a value greater than 0.1 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the solution is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a mean concentration of the therapeutic agent in the vitreous of the rabbit eye that is approximately constant at 1.0 ng/mL from 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.001 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.005 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.01 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 1.0 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 0.50 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 0.15 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 0.1 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.005 ng/mg and 1.0 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.005 ng/mg and 0.50 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.005 ng/mg and 0.15 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.005 ng/mg and 0.1 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 1.0 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected between the sclera and conjunctiva of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 0.50 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 0.15 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 0.1 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 100. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 50. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 10. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 5.

In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye that remains approximately constant at a value greater than 0.001 ng/mg for 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation is injected between the sclera and conjunctiva of a rabbit eye to deliver the therapeutic agent, resulting in a mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye that remains approximately constant at a value greater than 0.005 ng/mg for 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye, such that the average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye remains approximately constant at a value greater than 0.01 ng/mg from 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least 100 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least 1000 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least 10,000 ng/mL for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 100 ng/mL and 100,000 ng/mL within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 100 ng/mL and 50,000 ng/mL within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 1000 ng/mL and 100,000 ng/mL within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the vitreous of the rabbit eye of between 1000 ng/mL and 50,000 ng/mL within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye is less than 100. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye is less than 50. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the vitreous of the rabbit eye to the minimum mean concentration of the therapeutic agent in the vitreous of the rabbit eye is less than 10.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that the average concentration of the therapeutic agent in the vitreous of the rabbit eye remains approximately constant at a value greater than 100 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that the average concentration of the therapeutic agent in the vitreous of the rabbit eye remains approximately constant at a value greater than 1000 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that the average concentration of the therapeutic agent in the vitreous of the rabbit eye remains approximately constant at a value greater than 10,000 ng/mL for 30 days to at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation is injected into the vitreous of rabbit eyes to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.001 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eyes. In some variations, the liquid formulation is injected into the vitreous of rabbit eyes to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.01 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eyes. In some variations, the liquid formulation is injected into the vitreous of rabbit eyes to deliver the therapeutic agent, resulting in an average concentration of the therapeutic agent in the retinal choroidal tissue of at least 0.05 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eyes. In some variations, the liquid formulation delivers the therapeutic agent when injected into the vitreous of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of at least 0.10 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 10.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 5.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected into the vitreous of a rabbit eye to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.001 ng/mg and 1.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 10.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 5.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to achieve an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.01 ng/mg and 1.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.05 ng/mg and 10.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.05 ng/mg and 5.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.05 ng/mg and 1.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.10 ng/mg and 10.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.10 ng/mg and 5.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent to obtain an average concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye of between 0.10 ng/mg and 1.00 ng/mg for at least 30 days, at least 60 days, at least 90 days, or at least 120 days after the liquid formulation is administered to the rabbit eye.

In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 100. In some variations, the liquid formulation, when injected into the vitreous of a rabbit eye, delivers the therapeutic agent such that, within 30 days to at least 60 days, at least 90 days, or at least 120 days after administration of the liquid formulation to the rabbit eye, the ratio of the maximum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye to the minimum mean concentration of the therapeutic agent in the retinal choroidal tissue of the rabbit eye is less than 50.

In some variations, the in situ gelling formulations described herein can have an in vivo delivery profile to the retinal choroidal tissue characterized by delivery of the therapeutic agent in vivo following injection of the liquid formulation between the sclera and conjunctiva of a rabbit eye.

At 32 days after injection, the percent vitreous level in vivo can be between about 20% and about 80%, and more often between about 40% and about 60%, relative to the level expressed at day 7 after injection. At 40 days after injection, the percent vitreous level in vivo can be greater than about 20%, and more often greater than about 40%, relative to the level expressed at day 7 after injection.

At 45 days after injection, the percent vitreous level in vivo can be between about 15% and about 55%, and more often between about 25% and about 45%, relative to the level expressed at day 7 after injection. At 67 days after injection, the percent vitreous level in vivo can be greater than about 15%, and more often greater than about 25%, relative to the level expressed at day 7 after injection.

At 90 days after injection, the percent vitreous level in vivo can be between about 60% and about 100%, and more often between about 70% and about 90%, relative to the level expressed at day 7 after injection. At 90 days after injection, the percent vitreous level in vivo can be greater than about 60%, and more often greater than about 70%, relative to the level expressed at day 7 after injection.

In some variations, the in vivo percent vitreous level relative to the level at day 7 post-injection is characterized by: at day 32 post-injection, it is less than about 80%; at day 45 post-injection, it is less than about 60%; and at day 90 post-injection, it is less than about 100%.

In some variations, the liquid formulation is injected into the rabbit eye between the sclera and conjunctiva to deliver the therapeutic agent, resulting in an average concentration of at least about 0.1 pg/mg of the therapeutic agent in the rabbit eye retinal choroidal tissue within at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected into the rabbit eye between the sclera and conjunctiva to deliver the therapeutic agent, resulting in an average concentration of at least about 0.01 ng/mg of the therapeutic agent in the rabbit eye vitreous within at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation is injected into the rabbit eye between the sclera and conjunctiva to deliver the therapeutic agent, resulting in an average concentration of at least about 0.1 ng/mg of the therapeutic agent in the rabbit eye vitreous within at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is applied to the rabbit eye. In some variations, the liquid formulation delivers the therapeutic agent when injected between the sclera and conjunctiva of a rabbit eye to achieve an average concentration of the therapeutic agent in the vitreous of the rabbit eye of at least about 1 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye.

In some variations, after placement of the in situ gelling formulation in or near an eye, the logarithm base ten ratio of the average levels of the therapeutic agent in two or more of the retinal choroidal tissue, the sclera, and the vitreous remains approximately constant over an extended period of time. In some variations, after placement of the in situ gelling formulation between the sclera and conjunctiva of an eye, the logarithm base ten ratio of the average levels of the therapeutic agent in two or more of the retinal choroidal tissue, the sclera, and the vitreous remains approximately constant over an extended period of time. In some variations, after placement of the in situ gelling formulation between the sclera and conjunctiva of an eye, the logarithm base ten ratio of the average levels of the therapeutic agent in the vitreous and sclera remains approximately constant over an extended period of time.

In some variations, the ratio of the logarithm to the base 10 of the average levels of the therapeutic agent in the vitreous and retinal choroidal tissues is approximately constant over an extended period of time. In other words, when considered on a logarithmic scale, as the level of the therapeutic agent in the vitreous increases, the level of the therapeutic agent in the retinal choroidal tissue increases to a similar extent, and vice versa.

In some variations, the decadic logarithm of the ratio of the average level of therapeutic agent in the vitreous to the retinal choroidal tissue remains approximately constant over an extended period of about 7 days, about 30 days, about 60 days, or about 90 days. In some variations, after placement of the in situ gelling formulation between the sclera and conjunctiva of the eye, the ratio of the average level of therapeutic agent in the vitreous to the average level of therapeutic agent in the retinal choroidal tissue is constant at about 37:1 on day 7, about 40:1 on day 32, about 10:1 on day 45, and about 34:1 on day 90.

In some variations, the ratio of the average level of therapeutic agent in the vitreous relative to the average level of therapeutic agent in the retinal choroidal tissue is constant at about 40:1 at about 7 days, about 32 days, about 45 days, or about 90 days.

In some variations, after placement of the in situ gelling formulation in or near the eye, the average level of the therapeutic agent in any or all of the retinal choroidal tissue, sclera, and vitreous remains approximately unchanged for an extended period of time.

In some variations, after the in situ gelling formulation is positioned between the sclera and the conjunctiva, the average level of the therapeutic agent in the vitreous remains approximately constant at about 8.1 ng/ml. In some variations, after the in situ gelling formulation is positioned between the sclera and the conjunctiva, the average level of the therapeutic agent in the retinal choroidal tissue remains approximately constant at about 0.25 ng/mg. In some variations, after the in situ gelling formulation is positioned between the sclera and the conjunctiva, the average concentration of the therapeutic agent in the sclera remains approximately constant at about 1930 ng/mg.

In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains a mean level of the therapeutic agent in the vitreous of the rabbit eye at approximately 0.1 pg/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye. In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains a mean level of the therapeutic agent in the vitreous of the rabbit eye at approximately 0.001 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye. In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains a mean level of the therapeutic agent in the vitreous of the rabbit eye at approximately 0.01 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye. In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains an average level of the therapeutic agent in the vitreous of the rabbit eye at approximately 0.1 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains an average level of the therapeutic agent in the vitreous of the rabbit eye at approximately 1 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, the in situ gelling formulation, when injected between the sclera and conjunctiva of a rabbit eye, maintains an average level of the therapeutic agent in the vitreous of the rabbit eye at approximately 10 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, the in situ gelling formulation when injected between the sclera and conjunctiva of a rabbit eye maintains a substantially constant level of therapeutic agent in the vitreous of the rabbit eye at a mean level of about 100 ng/mL for at least about 30 days, at least about 60 days, or at least about 90 days after administration of the liquid formulation to the rabbit eye.

In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 0.1 pg/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 0.001 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 0.01 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 0.1 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 1 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the retinal choroidal tissue approximately constant at about 10 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye.

In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 0.1 pg/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 0.001 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 0.01 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 0.1 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 1 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 10 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 100 ng/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 1 μg/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye. In some variations, when injected between the sclera and conjunctiva of a rabbit eye, the in situ gelling formulation maintains an average level of the therapeutic agent in the sclera at approximately 10 μg/mg for at least about 30 days, at least about 60 days, or at least about 90 days after the liquid formulation is administered to the rabbit eye.

In order to treat, prevent, suppress, delay the occurrence of certain disease or illness or make it disappear, it is desirable to maintain the therapeutic agent of delivering therapeutically effective amount in the time period of extension.Based on the disease or illness that treats, prevents, suppresses, delays its occurrence or makes it disappear, the time period of this extension can be at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months or at least about 1 year.However, usually any extended delivery time can be possible.The therapeutic agent of therapeutically effective amount can be delivered the time of extension by liquid preparation or composition, and described liquid preparation or composition maintain the concentration of therapeutic agent in the subject or subject's eye within the time of extension, and described concentration is enough to deliver the therapeutic agent of therapeutically effective amount within the time of extension.

The therapeutic agent of sending treatment effective dose in the time of extension can be by placing a kind of compositions or liquid preparation, or by using the compositions or the liquid preparation of two or more dosage to complete.As the limiting examples of this type of repeatedly application, the therapeutic dose of rapamycin is maintained for three months to be used for the treatment of, prevention, inhibition, delay the generation of wet AMD or make it disappear and can be by using a kind of liquid preparation or the compositions of sending therapeutic dose for 3 months, or by sequentially using multiple liquid preparations or compositions to complete.The optimal dose strategy will depend on the therapeutic dose of the therapeutic agent that needs to be sent, and the time that it needs to be sent.The technical staff of extending therapeutic agent delivery administration field will understand how to determine spendable administration strategy based on this paper teaching.

When using certain therapeutic agents or for treating, preventing, suppressing, delaying the generation of certain diseases or making it disappear, it is desirable that the delivery of therapeutic agents does not start immediately when the liquid preparation or composition is placed in the eye region, but starts to deliver after a certain delay. For example, (but not limiting), when the therapeutic agent suppresses or delays wound healing, and needs the delayed release to make any wound healing produced when placing the liquid preparation or composition, this type of delayed release can be useful. According to the therapeutic agent being delivered and/or the disease and illness being treated, prevented, suppressed, delayed its generation or making it disappear, the delay period before the therapeutic agent delivery starts can be about 1 hour, about 6 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days or about 42 days. Other delay periods also may be possible. Delayed-release formulations that can be used are known to those skilled in the art.

Intravitreal and subconjunctival delivery of rapamycin for the treatment, prevention, inhibition, delay, or regression of AMD

In a method described herein, a liquid formulation comprising rapamycin is delivered subconjunctivally or intravitreally to treat, prevent, inhibit, delay, or regress angiogenesis in the eye, including but not limited to treating CNV as observed in AMD. In some variations, the liquid formulation is used to treat angiogenesis in the eye, including but not limited to treating CNV as observed in AMD. Rapamycin has been shown to inhibit CNV in rabbit and mouse models, as described in U.S. Application No. 10/665,203, which is incorporated herein by reference in its entirety. Rapamycin has been observed to inhibit Matrigel ^™ and laser-induced CNV when administered systemically and subretinal. Additionally, periocular injections of rapamycin inhibit laser-induced CNV.

Other therapeutic agents that can be delivered to the eye (particularly the vitreous) for treating, preventing, inhibiting, delaying the onset of, or regressing ocular angiogenesis (eg, CNV) are compounds of the limus family other than rapamycin, including but not limited to everolimus and tacrolimus (FK-506).

As described herein, the dosage of the therapeutic agent depends on the condition being treated (whether the condition is to be treated, prevented, inhibited, delayed in onset, or resolved), the specific therapeutic agent, and other clinical factors, such as the subject's weight and condition, and the route of administration of the therapeutic agent. It should be understood that the methods, liquid formulations, and compositions described herein can be used for both human and veterinary purposes, as well as for other possible animals. As described herein, tissue concentrations of therapeutic agents expressed in mass/volume units generally refer to substantially aqueous tissues, such as the vitreous. Tissue concentrations of therapeutic agents expressed in mass/mass units generally refer to other tissues, such as the sclera or retinal choroidal tissue.

One concentration of rapamycin that can be used in the methods described herein is one that provides a tissue level of rapamycin of about 0.01 pg/ml or pg/mg or more. Another concentration that can be used is one that provides a tissue level of about 0.1 pg/ml or ng/mg or more. Another concentration that can be used is one that provides a tissue level of about 1 pg/ml or ng/mg or more. Another concentration that can be used is one that provides a tissue level of about 0.01 ng/ml or ng/mg or more. Another concentration that can be used is one that provides a tissue level of about 0.1 ng/ml or ng/mg or more. Another concentration that can be used is one that provides a tissue level of about 0.5 ng/ml or ng/mg or more. Another concentration that can be used is one that provides a tissue level of about 1 ng/ml or more. Another concentration that can be used is one that provides a tissue level of about 2 ng/ml or more. Another concentration that can be used is one that provides a tissue level of about 3 ng/ml or more. Another concentration that can be used is one that provides a tissue level of about 5 ng/ml or more. Another concentration that can be used is to provide a concentration of about 10 ng/ml or more at the tissue level. Another concentration that can be used is to provide a concentration of about 15 ng/ml or more at the tissue level. Another concentration that can be used is to provide a concentration of about 20 ng/ml or more at the tissue level. Another concentration that can be used is to provide a concentration of about 30 ng/ml or more at the tissue level. Another concentration that can be used is to provide a concentration of about 50 ng/ml or more at the tissue level. Those skilled in the art will know how to achieve the appropriate concentration based on the route of administration used and the duration according to the teachings herein.

Generally, the amount of rapamycin administered in the liquid formulation is an amount sufficient to treat, prevent, inhibit, delay the onset of, or cause regression of an ocular disease or condition for the desired amount of time. In some variations, the amount of rapamycin administered in the liquid formulation is an amount sufficient to treat an ocular disease or condition for the desired amount of time.

In some variations, less than about 5 mg of rapamycin is administered subconjunctivally. In some variations, less than about 5.0 mg of rapamycin is administered subconjunctivally. In some variations, less than about 4.5 mg of rapamycin is administered subconjunctivally. In some variations, less than about 4.0 mg of rapamycin is administered subconjunctivally. In some variations, less than about 3.5 mg of rapamycin is administered subconjunctivally. In some variations, less than about 3.0 mg of rapamycin is administered subconjunctivally. In some variations, less than about 2.5 mg of rapamycin is administered subconjunctivally. In some variations, less than about 2 mg of rapamycin is administered subconjunctivally. In some variations, less than about 1.2 mg of rapamycin is administered subconjunctivally. In some variations, less than about 1.0 mg of rapamycin is administered subconjunctivally. In some variations, less than about 0.8 mg of rapamycin is administered subconjunctivally. In some variations, less than about 0.6 mg of total rapamycin is administered subconjunctivally. In some variations, less than about 0.4 mg of total rapamycin is administered subconjunctivally. In some variations, a volume of formulation comprising an amount of rapamycin described herein is administered.

In some variations, a liquid formulation containing a rapamycin concentration of between about 0.5% and about 6% by weight is administered to a human subject's conjunctiva by administering between about 0.1 μl and about 200 μl of a liquid formulation described herein. In some variations, a liquid formulation containing a rapamycin concentration of between about 0.5% and about 4% by weight is administered to a human subject's conjunctiva by administering between about 1 μl and about 50 μl of a liquid formulation described herein. In some variations, a liquid formulation containing a rapamycin concentration of between about 1.5% and about 3.5% by weight is administered to a human subject's conjunctiva by administering between about 1 μl and about 15 μl of a liquid formulation described herein. In some variations, a liquid formulation containing a rapamycin concentration of about 2% by weight is administered to a human subject's conjunctiva by administering between about 1 μl and about 15 μl of a liquid formulation described herein.

In some variations, a liquid formulation containing between about 0.2 μg and about 4 mg of rapamycin is administered to a human subject subconjunctiva by administering between about 0.1 μl and about 200 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 2 mg of rapamycin is administered to a human subject subconjunctiva by administering between about 0.1 μl and about 100 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 1 mg of rapamycin is administered to a human subject subconjunctiva by administering between about 1 μl and about 50 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 500 μg of rapamycin is administered to a human subject subconjunctiva by administering between about 1 μl and about 25 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 300 μg of rapamycin is administered subconjunctivally to a human subject by administering between about 1 μl and about 15 μl of a liquid formulation described herein.

In some variations, less than about 200 μg of rapamycin is administered intravitreally. In some variations, less than about 200 μg of rapamycin is administered intravitreally. In some variations, less than about 300 μg of rapamycin is administered intravitreally. In some variations, less than about 400 μg of rapamycin is administered intravitreally. In some variations, less than about 500 μg of rapamycin is administered intravitreally. In some variations, less than about 600 μg of rapamycin is administered intravitreally. In some variations, less than about 800 μg of rapamycin is administered intravitreally. In some variations, less than about 1 mg of rapamycin is administered intravitreally. In some variations, less than about 2 mg of rapamycin is administered intravitreally. In some variations, less than about 2.5 mg of rapamycin is administered intravitreally. In some variations, less than about 3 mg of rapamycin is administered intravitreally. In some variations, less than about 3.5 mg of total rapamycin is administered intravitreally. In some variations, less than about 4 mg of total rapamycin is administered intravitreally. In some variations, a volume of formulation containing an amount of rapamycin described herein is administered.

In some variations, a liquid formulation comprising a rapamycin concentration of between about 0.5% and about 6% by weight of the total weight is administered to the vitreous of a human subject by administering between about 0.1 μl and about 200 μl of a liquid formulation described herein. In some variations, a liquid formulation comprising a rapamycin concentration of between about 0.5% and about 4% by weight of the total weight is administered to the vitreous of a human subject by administering between about 1 μl and about 50 μl of a liquid formulation described herein. In some variations, a liquid formulation comprising a rapamycin concentration of between about 1.5% and about 3.5% by weight of the total weight is administered to the vitreous of a human subject by administering between about 1 μl and about 15 μl of a liquid formulation described herein. In some variations, a liquid formulation comprising a rapamycin concentration of about 2% by weight of the total weight is administered to the vitreous of a human subject by administering between about 1 μl and about 15 μl of a liquid formulation described herein.

In some variations, a liquid formulation containing between about 0.2 μg and about 4 mg of rapamycin is administered intravitreally to a human subject by administering between about 0.1 μl and about 200 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 2 mg of rapamycin is administered intravitreally to a human subject by administering between about 1 μl and about 100 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 1 mg of rapamycin is administered intravitreally to a human subject by administering between about 1 μl and about 50 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 500 μg of rapamycin is administered intravitreally to a human subject by administering between about 1 μl and about 25 μl of a liquid formulation described herein. In some variations, a liquid formulation containing between about 20 μg and about 300 μg of rapamycin is intravitreally administered to a human subject by administering between about 1 μl and about 15 μl of a liquid formulation described herein.

In some variations, a liquid formulation as described herein containing between about 1 μg and about 5 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 4 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 1.2 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 0.5 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 90 μg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 60 μg and about 120 μg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 100 μg and about 400 μg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 400 μg and about 1 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 1 mg and about 5 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 3 mg and about 7 mg of rapamycin is administered to a human subject for the treatment of wet AMD. In some variations, a liquid formulation as described herein containing between about 5 mg and about 10 mg of rapamycin is administered to a human subject for the treatment of wet AMD.

In some variations, a liquid formulation as described herein containing between about 1 μg and about 5 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 4 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 1.2 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 0.5 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 90 μg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 60 μg and about 120 μg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 100 μg and about 400 μg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 400 μg and about 1 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 1 mg and about 5 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 3 mg and about 7 mg of rapamycin is administered to a human subject for the prevention of wet AMD. In some variations, a liquid formulation as described herein containing between about 5 mg and about 10 mg of rapamycin is administered to a human subject for the prevention of wet AMD.

In some variations, a liquid formulation as described herein containing between about 1 μg and about 5 mg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 4 mg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a liquid formulation as described herein containing between about 20 μg and about 1.2 mg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 0.5 mg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a liquid formulation as described herein containing between about 10 μg and about 90 μg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a liquid formulation as described herein containing between about 60 μg and about 120 μg of rapamycin is administered to a human subject for the treatment of dry AMD. In some variations, a human subject is administered a dose of rapamycin containing between about 100 μg and about 400 μg for the treatment of dry AMD. In some variations, a human subject is administered a dose of rapamycin containing between about 400 μg and about 1 mg for the treatment of dry AMD. In some variations, a human subject is administered a dose of rapamycin containing between about 1 mg and about 5 mg for the treatment of dry AMD. In some variations, a human subject is administered a dose of rapamycin containing between about 3 mg and about 7 mg for the treatment of dry AMD. In some variations, a human subject is administered a dose of rapamycin containing between about 5 mg and about 10 mg for the treatment of dry AMD.

In some variations, a liquid formulation described herein containing between about 1 μg and about 5 mg of rapamycin is administered to a human subject for the treatment of angiogenesis, including but not limited to choroidal neovascularization. In some variations, an amount of rapamycin is administered to a human subject in an amount of between about 20 μg and about 4 mg; between about 20 μg and about 1.2 mg; an amount of rapamycin is administered to a human subject in an amount of between about 10 μg and about 0.5 mg for the treatment of wet AMD; between about 10 μg and 90 μg, between about 60 μg and 120 μg; between about 100 μg and 400 μg, between about 400 μg and 1 mg; in some variations, an amount of rapamycin is administered to a human subject in an amount of between about 1 mg and 5 mg; in some variations, an amount of rapamycin is administered to a human subject in an amount of between about 3 mg and 7 mg; in some variations, an amount of rapamycin is administered to a human subject in an amount of between about 5 mg and 10 mg for the treatment of angiogenesis, including but not limited to choroidal neovascularization.

In one approach, the liquid formulations described herein contain an amount of therapeutic agent equivalent to an amount of rapamycin.

In one method, a liquid formulation described herein is administered to a human subject in an amount of a therapeutic agent equivalent to an amount of rapamycin between about 1 μg and about 5 mg for the treatment of wet AMD. In some variations, an amount of the therapeutic agent equivalent to between about 1 μg and about 5 mg of rapamycin is administered to a human subject; between about 20 μg and about 1.2 mg; between about 10 μg and about 0.5 mg is administered to a human subject for treating wet AMD, between about 10 μg and 90 μg, between about 60 μg and 120 μg is administered to a human subject; between about 100 μg and 400 μg, between about 400 μg and 1 mg is administered to a human subject; in some variations, an amount of the therapeutic agent equivalent to between about 1 mg and 5 mg of rapamycin is administered to a human subject; in some variations, an amount of the therapeutic agent equivalent to between about 3 mg and 7 mg of rapamycin is administered to a human subject; in some variations, an amount of the therapeutic agent equivalent to between about 5 mg and 10 mg of rapamycin is administered to a human subject.

In some variations, a liquid formulation described herein containing an amount of a therapeutic agent equivalent to an amount of rapamycin between about 1 μg and about 5 mg is administered to a human subject for the treatment of dry AMD. In some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 20 μg and about 4 mg is administered to a human subject; between about 20 μg and about 1.2 mg; between about 10 μg and about 0.5 mg is administered to a human subject. For the treatment of wet AMD, between about 10 μg and 90 μg, between about 60 μg and 120 μg are administered to a human subject; between about 100 μg and 400 μg, between about 400 μg and 1 mg are administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 400 μg and 1 mg is administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 1 mg and 5 mg is administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 10 μg and 90 μg, between about 60 μg and 120 μg are administered to a human subject; An amount of the therapeutic agent equivalent to an amount of rapamycin between 3 mg and 7 mg; in some variations, an amount of the therapeutic agent equivalent to an amount of rapamycin between about 5 mg and 10 mg is administered to a human subject to treat dry AMD.

In some variations, a liquid formulation described herein containing an amount of a therapeutic agent equivalent to an amount of rapamycin between about 1 μg and about 5 mg is administered to a human subject for the prevention of wet AMD. In some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 20 μg and about 4 μg is administered to a human subject; between about 20 μg and about 1.2 mg; between about 10 μg and about 0.5 mg is administered to a human subject. For the prevention of wet AMD, between about 10 μg and 90 μg, between about 60 μg and 120 μg are administered to a human subject; between about 100 μg and 400 μg, between about 400 μg and 1 mg are administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 400 μg and 1 mg is administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 1 mg and 5 mg is administered to a human subject; in some variations, an amount of a therapeutic agent equivalent to an amount of rapamycin between about 10 μg and 90 μg is administered to a human subject; between about 100 μg and 400 μg, between about 400 μg and 1 mg is administered to a human subject; An amount of the therapeutic agent equivalent to an amount of rapamycin between 3 mg and 7 mg; in some variations, an amount of the therapeutic agent equivalent to an amount of rapamycin between about 5 mg and 10 mg is administered to a human subject for preventing wet AMD.

In some modifications, every 3 or more months, every 6 or more months, every 9 or more months or every 12 or more months or longer time intravitreal administration of any one or more preparations described herein, in order to treat one or more in choroidal neovascularization, wet AMD, dry AMD, in order to prevent wet AMD or for preventing the development of dry AMD to wet AMD. In some modifications, every 3 or more months, every 6 or more months, every 9 or more months or every 12 or more months or longer time subconjunctival administration of any one or more preparations described herein, in order to treat one or more in choroidal neovascularization, wet AMD, dry AMD, or for preventing wet AMD.

In some modifications, any one or more rapamycin preparations described herein are administered intravitreally every 3 or more months, every 6 or more months, every 9 or more months, or every 12 or more months, or longer, to treat one or more of choroidal neovascularization, wet AMD, or dry AMD, to prevent wet AMD or to prevent the development of dry AMD to wet AMD. In some modifications, any one or more rapamycin preparations described herein are administered subconjunctivally every 3 or more months, every 6 or more months, every 9 or more months, or every 12 or more months, or longer, to treat one or more of choroidal neovascularization, wet AMD, or dry AMD, or to prevent wet AMD. In some modifications, the effect of rapamycin continues beyond the period during which it is present in ocular tissue.

The delivery of therapeutic agents described herein can be, for example, delivered in a dosage range between about 1 ng/ day and about 100 μg/ day, or in a dosage range above or below this range, depending on the route and duration of administration. In some variations of the liquid formulations or compositions used in the methods described herein, therapeutic agents can be delivered in a dosage range between about 0.1 μg/ day and about 10 μg/ day. In some variations of the liquid formulations or compositions used in the methods described herein, therapeutic agents can be delivered in a dosage range between about 1 μg/ day and about 5 μg/ day. The dosage of the various therapeutic agents for treating, preventing, suppressing, delaying the occurrence of, or causing the disappearance of, various diseases and conditions described herein can be optimized using clinical trials.

Liquid formulations, including but not limited to solutions, suspensions, emulsions, and in situ gelling formulations, and the compositions described herein, can be used to deliver a therapeutically effective amount of rapamycin to the eye over an extended period of time (as a non-limiting example, by ocular administration or periocular administration) for treating, preventing, inhibiting, delaying the onset of, or causing regression of CNV, and thus can be used to treat, prevent, inhibit, delay the onset of, or causing regression of wet AMD or the conversion of dry AMD to wet AMD. It is believed that by varying certain characteristics of the liquid formulations described herein (including but not limited to the ingredients of the liquid formulation, the location in the eye to which the liquid formulation is delivered, including but not limited to subconjunctival or intravitreal placement), the liquid formulations can be used to deliver a therapeutically effective amount of rapamycin to the eye over a variety of extended periods of time, including delivering a therapeutically effective amount for more than about 1 week, more than about 2 weeks, more than about 3 weeks, more than about 1 month, more than about 3 months, more than about 6 months, more than about 9 months, or more than about 1 year.

When a therapeutically effective amount of rapamycin is administered to a subject suffering from wet AMD, rapamycin can treat, inhibit, or regress wet AMD. Treating, inhibiting, or regressing wet AMD may require different therapeutically effective amounts. A subject suffering from wet AMD may have CNV lesions, and it is believed that administering a therapeutically effective amount of rapamycin may have various effects, including but not limited to causing regression of CNV lesions, stabilizing CNV lesions, and preventing the development of active CNV lesions.

When a therapeutically effective amount of rapamycin is administered to a subject with dry AMD, it is believed that rapamycin can prevent or delay the progression of dry AMD to wet AMD.

Example

Unless otherwise indicated by the context, error bars in the graphs represent one standard deviation. When ethanol was used, it was 200 proof ethanol from Gold Shield Distributors, Hayward, CA. When rapamycin was used, it was from LC laboratories, Woburn, MA or Chunghwa Chemical Synthesis & Biotech Co., LTD (CCSB), Taipei Hsien, Taiwan, ROC. When PEG400 was used, it was from The Dow Chemical Company, New Milford, CT. Some figures use the expression "uL" or "ug" to refer to μL or μL, respectively. When administering volumes of 10 μL or less, a Hamilton HPLC syringe was used.

Example 1 - Preparation and Characterization of Rapamycin-Containing Solutions

Dissolve 1.256% rapamycin (by weight) in 9.676% ethanol (by weight). Slowly add a 15% solution of Lutrol F127 (Lutrol) in sterile water with continuous stirring. The final concentration is approximately 78.57% sterile water (by weight) and approximately 10.50% Lutrol F127 (by weight). This solution is listed in Table 1 as Formulation #32. Keep the solution at 2°C until use.

Example 2 - Subconjunctival injection of a solution containing rapamycin

50 μl of the solution described in Example 1 were injected between the sclera and conjunctiva of New Zealand white rabbits.

Figure 2 depicts the mean concentrations of rapamycin present in the vitreous (ng/ml), retina choroid (ng/mg), and sclera (ng/mg) on a logarithmic scale at 20, 40, 67, and 90 days after injection.

Analysis was performed by liquid chromatography mass spectrometry (LCMS) using an internal standard.

At each time point, the mean rapamycin concentration was calculated by adding the rapamycin concentrations obtained for each eye of each rabbit and dividing by the total number of eyes analyzed. In this experiment, each time point represents the mean of both eyes of two rabbits (four eyes at that time point) or the mean of both eyes of one rabbit (two eyes at that time point).

The entire vitreous was homogenized and analyzed. The mean vitreous concentration was calculated by dividing the measured mass of rapamycin by the volume of vitreous analyzed. The samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered into the vitreous by the solution.

The mean levels of rapamycin in the vitreous were approximately 4.425, 3.800, 4.100 and 1.500 ng/ml 20, 40, 67 and 90 days after subconjunctival injection, respectively.

The entire retina and choroid were homogenized and analyzed. The mean retinal choroid concentration was calculated by dividing the measured mass of rapamycin by the mass of retinal choroid analyzed. The sample did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid by the solution.

The mean levels of rapamycin in the retina and choroid were approximately 0.055, 0.209, 0.080, and 0.017 ng/mg 20, 40, 67, and 90 days after subconjunctival injection, respectively.

The sclera was analyzed in the same manner as the retina and choroid. The sclera sample included the injection site; therefore, this measurement indicated the clearance of rapamycin from the sclera.

The mean intrascleral rapamycin levels were approximately 0.141, 0.271, 0.067, and 0.192 ng/ml 20, 40, 67, and 90 days after subconjunctival injection, respectively.

Example 3 - Preparation and Characterization of Rapamycin-Containing Solutions

5.233% rapamycin (percentage of the total weight of the formulation after all ingredients are added) was dissolved in 0.4177 g of EtOH; the amount of EtOH was reduced to 0.1296 g (6.344% w/w) by forced evaporation (heating). PEG 400 was added with continuous stirring. The final concentrations, as a percentage of the total weight, were approximately: 5.233% rapamycin, 6.344% ethanol, and 88.424% PEG 400. Upon contact with the vitreous, the formulation formed a non-dispersed mass relative to the surrounding medium. This solution is listed in Table 1 as Formulation #34.

Example 4 - Subconjunctival injection of a solution containing rapamycin

25 μl of the solution described in Example 3 were injected between the sclera and conjunctiva of New Zealand white rabbits.

Figure 3 depicts rapamycin levels present in the vitreous (ng/ml), retina choroid (ng/mg), and sclera (ng/mg) on a logarithmic scale at 14, 35, 62, and 85 days after injection.

The vitreous was homogenized and analyzed as described in Example 2, except that a single eye from each of three rabbits was analyzed on day 2; both eyes from each of two rabbits were analyzed on day 14; both eyes from one rabbit were analyzed on day 35; both eyes from one rabbit were analyzed on day 62; and a single eye from one rabbit and both eyes from another rabbit were analyzed on day 85.

vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous by the solution. The average level of rapamycin in the vitreous was approximately 3.57, 53.65, 9.00, 4.700, and 0.600 ng/ml 2, 14, 35, and 85 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, with samples taken on the same days as described above for the vitreous. Day 2 analysis was not performed. The retina and choroid samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid by the solution. The mean level of rapamycin in the retina and choroid was approximately 0.4815, 1.725, 0.057, and 0.009 ng/mg 14, 35, 62, and 85 days after subconjunctival injection, respectively.

Scleral samples were analyzed as described in Example 2, with samples taken on the same days as described for the retina and choroid. The scleral samples included the site of administration; therefore, this measurement indicated the clearance of rapamycin from the sclera. The mean level of rapamycin in the sclera was approximately 34.5815, 0.135, 0.042, and 0.163666667 ng/mg 14, 35, 62, and 85 days after subconjunctival injection, respectively.

Example 5 - Intravitreal injection of a solution containing rapamycin

25 μl of the solution described in Example 3 was injected into the vitreous of New Zealand white rabbit eyes. FIG4 depicts the logarithmic scale levels of rapamycin present in the vitreous (ng/ml), retina choroid (ng/mg), and sclera (ng/mg) at 14, 35, 62, and 90 days after injection. Also shown are the levels of rapamycin present in the vitreous (ng/ml) 2 days after injection.

The vitreous was homogenized and analyzed as described in Example 2, except that approximately 1 μl was analyzed from a single eye of each of three rabbits on day 2; from both eyes of each of two rabbits on day 14; from both eyes of one rabbit on day 35; from both eyes of one rabbit on day 62; and from both eyes of each of two rabbits on day 90.

With the exception of the Day 2 sample, vitreous samples included the application site. Administered solutions were avoided whenever possible. However, the accuracy of the measured rapamycin levels may be affected by sampling error due to inadvertent inclusion of administered solutions.

The average levels of rapamycin in the vitreous were approximately 11.4, 136538, 2850.3, 21820.35 and 27142.75 ng/ml 2, 14, 35, 62 and 90 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, with samples taken on the same days as for the vitreous. Day 2 analysis was not performed. The retina and choroid samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid via the solution. The mean level of rapamycin in the retina and choroid was approximately 5.78975, 244.485, 0.105, and 1.782 ng/mg 14, 35, 62, and 90 days after intravitreal injection, respectively.

Scleral samples were analyzed as described in Example 2, taken on the same days as for the retina and choroid. Scleral samples did not include the injection site; therefore, this measurement indicated the clearance of rapamycin from the sclera. The mean level of rapamycin in the sclera was approximately 0.5695, 12.34, 0.8505, and 0.71175 ng/mg 14, 35, 62, and 90 days after intravitreal injection, respectively.

Example 6 - Preparation and Characterization of Rapamycin-Containing Suspensions

6% rapamycin (percentage of total weight) was dispersed in 94% PEG 400 (percentage of total weight). This suspension is listed in Table 1 as Formulation #55.

Example 7 - Intravitreal injection of a rapamycin-containing suspension

The solution prepared in Example 6 was injected intravitreally into the eyes of New Zealand white rabbits. FIG5 depicts images of rabbit eyes injected intravitreally with 10 μl ( FIG5A ), 20 μl ( FIG5B ), and 40 μl ( FIG5C ) of 6% rapamycin suspended in PEG400. This resulted in injected doses of approximately 0.6, approximately 1.2, and approximately 2.4 mg. The images are focused on the administered suspension. These images show that the suspension formed a non-dispersed mass relative to the surrounding vitreous medium.

Example 8 - Preparation and Characterization of In Situ Gelling Formulations Containing Rapamycin

A liquid formulation of 4.2% rapamycin (obtained from LC laboratories in Woburn, MA and Chunghwa Chemical Synthesis & BioTech. Co, Ltd in Taiwan), 4.3% ethanol (obtained from Gold Shield Chemical in Hayward, CA), 2.2% PVPK90 (obtained from BASF), 87.1% PEG400 (obtained from DOW Chemical) and 2.2% Eudragit RL100 (obtained from Rohm Pharma Polymers), wherein all percentages are percentages based on total weight.

Dissolve Eudragit RL100 in ethanol. This step may require sonication and heating. Add the ethanol-Eudragit to the PEG400. Slowly add PVP to the Eudragit-ethanol-PEG solution to create a uniformly mixed solution. This step may require thorough mixing.

Add rapamycin and dissolve it in the Eudragit-ethanol-PEG-PVP mixture. Heat and sonication may be used. Mix the formulation thoroughly (using a vortexer or mixer) to achieve homogeneity. This formulation is listed as #37 in Table 1.

When placed in deionized water or tap water, the liquid formulation formed a non-dispersible mass. The non-dispersible mass appeared as a gel-like substance.

Example 9 - Subconjunctival injection of a non-dispersed mass of rapamycin-containing formulation

50 μl of the solution described in Example 8 were injected between the sclera and conjunctiva of New Zealand white rabbits.

Figure 6 depicts the mean concentrations of rapamycin in the vitreous (ng/ml), retina and choroid (ng/mg), and sclera (ng/mg) 7, 32, 45, and 90 days after injection of the in situ gelling formulation.

Analyzed by LCMS (liquid chromatography-mass spectrometry).

When more than one eye was analyzed, the mean rapamycin concentration was calculated by adding the rapamycin concentrations from each eye of each rabbit and dividing the total by the number of eyes analyzed. In this experiment, the vitreous day 7 and scleral day 7, 32, and 45 time points were representative of a single eye relative to the mean level. The remaining day 7, 32, and 45 time points were representative of the mean values for both eyes of one rabbit, while the day 90 time point was representative of the mean values for both eyes of each of two rabbits (four eyes total).

The entire vitreous was homogenized and analyzed. The mean vitreous concentration was calculated by dividing the measured mass of rapamycin by the vitreous volume analyzed. The sample did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the vitreous by the in situ gelling formulation.

The mean levels of rapamycin in the vitreous were approximately 13.9, 7.4, 1.35, and 90 days after subconjunctival injection, respectively.

The entire retina was homogenized and analyzed. The mean retinal choroidal concentration was calculated by dividing the measured mass of rapamycin by the mass of retinal choroidal tissue analyzed. The sample did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retinal choroidal tissue by the in situ gelling formulation.

The average levels of rapamycin in the retinal choroidal tissue were approximately 0.376, approximately 0.1875, approximately 0.136, and approximately 0.29 ng/mg 7, 32, 45, and 90 days after subconjunctival injection, respectively.

The sclera is analyzed in the same manner as the retinal choroidal tissue. The sclera sample may include the injected liquid formulation; therefore, this measurement indicates the clearance rate of rapamycin from the sclera.

The mean levels of rapamycin in the sclera were approximately 2033, approximately 1653, approximately 3626, and approximately 420.5 ng/mg 7, 32, 45, and 90 days after subconjunctival injection, respectively.

Example 10 - Preparation and Characterization of Rapamycin-Containing Suspensions

A rapamycin suspension was prepared by dispersing 150.5 mg of rapamycin (3.004% by weight) in 4860.3 mg of PEG 400 (96.996% by weight). This formulation is listed as #49 in Table 1. 150.5 mg of rapamycin (3.004% by weight) and 4860.3 mg of PEG 400 (96.996% by weight) were placed in an amber tube. 3 mm diameter High Wear Resistant Zirconia Grinding Media (beads) was added up to three quarters of the total volume. The tube was sealed and placed in a Cole-Parmer grinding apparatus for 48 hours. The median rapamycin particle size was 2.8386 mm and the mean was 3.1275 mm. The formulation was stored at 4°C until use. Volumes of 20 μl and 40 μl each formed non-dispersed clumps when placed in the vitreous of a rabbit eye.

Example 11 - Subconjunctival injection of a suspension containing rapamycin

40 μl of the suspension described in Example 10 was injected between the sclera and conjunctiva of New Zealand white rabbits. Figure 7 depicts logarithmic scale levels of rapamycin in the vitreous (ng/ml), retina choroid (ng/mg) and sclera (ng/mg) 14, 42, 63 and 91 days after injection.

The vitreous was homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point, except on day 91, when both eyes from one rabbit were analyzed. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The mean levels of rapamycin in the vitreous were approximately 4.031, 23.11, 53.27, and 13.94 ng/ml at 14, 42, 63, and 91 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, with samples taken on the same days as for the vitreous. The retina and choroid do not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.1577, 4.965, 0.385, and 0.05 ng/mg 14, 42, 63, and 91 days after subconjunctival injection, respectively.

Scleral samples were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The scleral samples included the injection site. The average level of rapamycin in the sclera was approximately 1283, 476.3, 854.2, and 168.5 ng/mg 14, 42, 63, and 91 days after subconjunctival injection, respectively.

Example 12 - Intravitreal injection of a rapamycin-containing suspension

20 μl of the suspension described in Example 10 was injected into the vitreous of New Zealand white rabbit eyes. The injected suspension formed a non-dispersed mass relative to the surrounding medium. FIG8 depicts rapamycin levels in the retina, choroid (ng/mg) and sclera (ng/mg) at 14, 42, 63, and 91 days after injection, and in the vitreous (ng/ml) at 63 and 91 days after injection on a logarithmic scale.

The vitreous was homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point. The vitreous samples may contain the site of administration. The average level of rapamycin in the vitreous was approximately 381,600 and 150,400 ng/ml 63 and 91 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point. The retina and choroid do not include the site of administration, so this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 2.588, 4.249, 21.42, and 0.922 ng/mg 14, 42, 63, and 91 days after intravitreal injection, respectively.

Scleral samples were homogenized and analyzed as described in Example 2, and were sampled as described above for the retina and choroid. The scleral samples did not include the injection site; therefore, this measurement indicated the level of rapamycin delivered to the sclera. The average level of rapamycin in the sclera was approximately 0.7327, 6.053, 1.373, and 17.49 ng/mg 14, 42, 63, and 91 days after intravitreal injection, respectively.

Example 13 - Preparation and Characterization of Rapamycin-Containing Solutions

A rapamycin-containing solution was formed by dissolving 116.6 mg of rapamycin in ethanol and storing the mixture at 4°C for 6 hours. This solution was then mixed with 4647.5 mg of PEG 400 to yield a solution having a final concentration of 2.29% rapamycin, 6.05% ethanol, and 91.66% PEG 400 by weight. This solution is listed as Formulation #51 in Table 1. A volume of 30 μl formed a non-dispersed mass when placed in the vitreous of a rabbit eye.

Example 14 - Subconjunctival injection of a solution containing rapamycin

40 μl of the solution described in Example 13 was injected between the sclera and conjunctiva of New Zealand white rabbit eyes. Figure 9 depicts rapamycin levels in the vitreous (ng/ml), retina choroid (ng/mg) and sclera (ng/mg) on a linear scale at 14, 42, 63 and 91 days after injection.

The vitreous was homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point, except on day 91, when both eyes from one rabbit were analyzed. The vitreous samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered into the vitreous. The mean level of rapamycin in the vitreous was approximately 1.804, 1.854, 1.785, and 1.255 ng/ml at 14, 42, 63, and 91 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 1.221, 4.697, 0.1075, and 0.02 ng/mg 14, 42, 63, and 91 days after subconjunctival injection, respectively.

Scleral samples were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The scleral samples included the injection site. The mean level of rapamycin in the sclera was approximately 1.987, 1.884, 0.56, and 10.84 ng/mg 14, 42, 63, and 91 days after subconjunctival injection, respectively.

Example 15 - Intravitreal injection of a solution containing rapamycin

30 μl of the solution described in Example 13 was injected into the vitreous of New Zealand white rabbit eyes. The injected solution formed a non-dispersed mass relative to the surrounding medium. Figure 10 depicts rapamycin levels in the retina, choroid (ng/mg) and sclera (ng/mg) on a linear scale at 14, 42, 63, and 91 days after injection.

The retina and choroid were homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point. The retina and choroid do not include the site of administration, so this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 5.515, 5.388, 0.3833, and 11.52 ng/mg 14, 42, 63, and 91 days after intravitreal injection, respectively.

Scleral samples were homogenized and analyzed as described in Example 2 and sampled as described above for the retina and choroid. The scleral samples did not include the injection site, so this measurement indicated the level of rapamycin delivered to the sclera. The average level of rapamycin in the sclera was approximately 1.077, 0.9239, 0.0975, and 2.0825 ng/mg 14, 42, 63, and 91 days after intravitreal injection, respectively.

FIG11 depicts the linear scale of rapamycin levels in the vitreous (ng/ml) 63 and 91 days after injection. The vitreous was homogenized and analyzed as described in Example 2. Both eyes from two rabbits were analyzed at each time point. The vitreous samples may contain the site of administration. The average level of rapamycin in the vitreous 63 and 91 days after intravitreal injection was approximately 299,900 and 196,600 ng/ml, respectively.

Example 16 - Preparation and Characterization of Rapamycin-Containing Solutions

About 320 g of ethanol was flushed with N2 for about 10 minutes, and then about 40 g of sirolimus was added to the ethanol. The mixture was sonicated for about 20 minutes, at which point all of the sirolimus had entered the solution, forming a sirolimus stock solution. A dilution solvent was prepared by sonicating about 1880 g of PEG400 for about 60 minutes, and then flushing the solvent with nitrogen for about 10 minutes.

The sirolimus stock solution and PEG 400 were then rotated in a rotary evaporator at approximately room temperature for approximately 10 minutes to mix the stock solution and the dilution solvent. After mixing, the solution was flushed with nitrogen for approximately 10 minutes and blanketed with nitrogen for approximately 5 minutes. Once the solution was flushed and filled with nitrogen, approximately 240 g of excess ethanol was evaporated from the solution by increasing the temperature of the solution, maintaining the temperature at no more than 40° C. for an extended period of time, and continuing to rotate the solution for approximately 2.5 hours.

The resulting solution contained approximately 40 g of sirolimus (approximately 2% by weight), approximately 80 g of ethanol (approximately 4% by weight), and approximately 1880 g of PEG400 (approximately 94% by weight). The solution was flushed with nitrogen for approximately 10 minutes and blanketed with nitrogen for approximately 5 minutes. The solution was then filtered through a 0.2 micron filter. 2 ml of each filtered solution was filled into HPLC vials, leaving approximately 400 μl of headspace in each container. The headspace was filled with nitrogen and capped.

Example 17 - Preparation and Characterization of Rapamycin-Containing Solutions

Rapamycin, ethanol, and PEG 400 were placed in a container to achieve a final concentration of approximately 2.00% rapamycin, approximately 4.00% ethanol, and approximately 94.00% PEG 400 by weight. The mixture was capped and sonicated for 1-2 hours. Sonication generates heat, reaching temperatures of approximately 40 or 50°C. This solution is listed as Formulation #100 in Table 1. Volumes of 1 μl, 3 μl, 20 μl, and 40 μl formed non-dispersed clumps in the vitreous of rabbit eyes.

Example 18 - Subconjunctival injection of a solution containing rapamycin

20 μl of the solution described in Example 17 was injected between the sclera and conjunctiva of New Zealand white rabbits. Figure 12 depicts the logarithmic scale of rapamycin levels present in the vitreous at 5, 30, 60, 90, and 120 days after injection. Figure 13 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points. For comparison, Figures 12 and 13 also depict the results of similar studies conducted with 40 μl and 60 μl injections, described below in Examples 19 and 20.

In Figures 12-15 discussed in this example and the following examples, some outliers have been omitted. Individual data points from the same study at the same time point were compared with each other. Data points were considered outliers when their arithmetic mean was lower than their standard deviation, either higher or lower than an order of magnitude.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 1.81, 0.45, 0.39, 1.85, and 1.49 ng/ml at 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.14, 0.03, 0.02, 0.02, and 0.01 ng/mg 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

Example 19 - Subconjunctival injection of a solution containing rapamycin

40 μl of the solution described in Example 17 was injected between the sclera and conjunctiva of New Zealand white rabbits. Figure 12 depicts the logarithmic scale of rapamycin levels present in the vitreous at 5, 30, 60, 90 and 120 days after injection. Figure 13 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 2.39, 0.65, 0.54, 2.07, and 1.92 ng/ml at 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.47, 0.04, 0.01, 0.05, and 0.0 ng/mg 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

Example 20 - Subconjunctival injection of a solution containing rapamycin

60 μl of the solution described in Example 17 was injected between the sclera and conjunctiva of New Zealand white rabbits. Figure 12 depicts the logarithmic scale of rapamycin levels present in the vitreous at 5, 30, 60, 90 and 120 days after injection. Figure 13 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 8.65, 0.29, 0.18, 2.00, and 1.41 ng/ml at 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.63, 0.02, 0.02, 0.06, and 0.01 ng/mg 5, 30, 60, 90, and 120 days after subconjunctival injection, respectively.

Example 21 - Intravitreal injection of a solution containing rapamycin

20 μl of the solution described in Example 17 was injected into the vitreous of New Zealand white rabbit eyes. The injected solution formed a non-dispersed mass relative to the surrounding medium. FIG14 depicts logarithmic rapamycin levels in the vitreous at 5, 30, 60, 90, and 120 days after injection. FIG15 depicts logarithmic rapamycin levels in the retina and choroid at the same time points. For comparison, FIG14 and FIG15 also depict the results of additional studies described in Examples 22 and 24 below.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. Vitreous samples may include the site of administration. Mean intravitreal rapamycin levels at 5, 30, 60, 90, and 120 days after intravitreal injection were approximately 162,100; 18,780; 57,830; 94,040; and 13,150 ng/ml, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 2.84, 2.26, 0.17, 0.22, and 0.05 ng/mg 5, 30, 60, 90, and 120 days after intravitreal injection, respectively.

Example 22 - Intravitreal injection of a solution containing rapamycin

40 μl of the solution described in Example 17 was injected into the vitreous of New Zealand white rabbits. The injected solution formed a non-dispersed mass relative to the surrounding medium. FIG14 depicts logarithmic rapamycin levels in the vitreous at 5, 30, 60, 90, and 120 days after injection. FIG15 depicts logarithmic rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. Vitreous samples may include the site of administration. Mean intravitreal rapamycin levels were approximately 415,600, 4,830, 74,510, 301,300, and 7,854 ng/ml at 5, 30, 60, 90, and 120 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 5.36, 0.23, 1.27, 1.08, and 0.08 ng/mg 5, 30, 60, 90, and 120 days after intravitreal injection, respectively.

Example 23 - Preparation and Characterization of Rapamycin-Containing Solutions.

Rapamycin, ethanol, and PEG 400 were placed in a container to achieve a final concentration of approximately 0.4% rapamycin, approximately 4.0% ethanol, and approximately 95.6% PEG 400 by weight. The mixture was sonicated for 1-2 hours. Sonication caused the temperature to rise to a maximum of approximately 40 to 50°C. This solution is listed as Formulation #99 in Table 1.

Example 24 - Intravitreal injection of a solution containing rapamycin

100 μl of the solution described in Example 23 was injected into the vitreous of New Zealand white rabbit eyes. The injected solution did not form non-dispersed clumps relative to the surrounding medium. FIG14 depicts logarithmic-scale rapamycin levels in the vitreous at 5, 30, 60, 90, and 120 days after injection. FIG15 depicts logarithmic-scale rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Two to five rabbit eyes were analyzed at each time point. Vitreous samples may include the site of administration. Mean levels of rapamycin in the vitreous were approximately 151,000, 14,890, 4,743, and 1620 ng/ml at 5, 30, 60, 90, and 120 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 1.21, 1.84, 0.04, 0.71, and 0.0 ng/mg at 5, 30, 60, 90, and 120 days after intravitreal injection, respectively.

Example 25 Preparation and Characterization of Rapamycin-Containing Solutions.

A rapamycin-containing solution was formed by dissolving 102.4 mg of rapamycin in ethanol, adding 4719.3 mg of PEG 400, and vortexing. The resulting solution had a final concentration of 2.036% rapamycin, 4.154% ethanol, and 93.81% PEG 400 by weight. This solution is listed in Table 1 as Formulation #139.

Example 26 Subconjunctival injection of a solution containing rapamycin

Ten microliters of the solution described in Example 25 were injected as a single dose between the sclera and conjunctiva of New Zealand white rabbits. Figure 16 depicts the logarithmic scale of rapamycin levels in the vitreous at 5 and 14 days after injection. Figure 17 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points. For comparison, Figures 16 and 17 also depict the results of other studies described in Examples 27-29 below.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 2.45 and 20.13 ng/ml at 5 and 14 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.13 and 0.19 ng/mg 5 and 14 days after subconjunctival injection, respectively.

Example 27 - Subconjunctival injection of a solution containing rapamycin

60 μl of the solution described in Example 25 was injected as a single dose between the sclera and conjunctiva of New Zealand white rabbits. Figure 16 depicts the logarithmic scale of rapamycin levels present in the vitreous 5 and 14 days after injection. Figure 17 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 17.98 and 87.03 ng/ml at 5 and 14 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.27 and 0.21 ng/mg, respectively, 5 and 14 days after subconjunctival injection.

Example 28 - Subconjunctival injection of a solution containing rapamycin

60 μl of the solution described in Example 25 was injected as two 30 μl doses into two sites between the sclera and conjunctiva of New Zealand white rabbits. Figure 16 depicts the logarithmic scale of rapamycin levels present in the vitreous 5 and 14 days after injection. Figure 17 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 502.2 and 31.80 ng/ml at 5 and 14 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.80 and 0.15 ng/mg, respectively, 5 and 14 days after subconjunctival injection.

Example 29 - Subconjunctival injection of a solution containing rapamycin

90 μl of the solution described in Example 25 was injected as three 30 μl doses into three sites between the sclera and conjunctiva of New Zealand white rabbits. Figure 16 depicts the logarithmic scale of rapamycin levels present in the vitreous 5 and 14 days after injection. Figure 17 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 39.05 and 13.63 ng/ml at 5 and 14 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.83 and 0.10 ng/mg, respectively, 5 and 14 days after subconjunctival injection.

Example 30 - Preparation and Characterization of Rapamycin-Containing Suspensions

A rapamycin suspension was prepared by disposing 1201.6 mg of rapamycin (3.000% by weight) in 6518.8 mg of PEG 400 (97.000% by weight) and vortexing. The resulting particle size was not quantified but was large, estimated to be approximately 10 μm. This suspension is listed as Formulation #147 in Table 1.

Example 31 - Subconjunctival injection of a suspension containing rapamycin

Ten μl of the suspension described in Example 30 was injected as a single dose between the sclera and conjunctiva of New Zealand white rabbits. Figure 18 depicts the logarithmic scale of rapamycin levels in the vitreous at 5, 14, and 30 days after injection. Figure 19 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points. For comparison, Figures 18 and 19 also depict the results of other studies described in Examples 32 and 33 below.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 2.68, 0.90, and 5.43 ng/ml at 5, 14, and 30 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.20, 0.06, and 1.23 ng/mg 5, 14, and 30 days after subconjunctival injection, respectively.

Example 32 - Subconjunctival injection of a suspension containing rapamycin

30 μl of the solution described in Example 30 was injected as a single dose into the sclera and conjunctiva of New Zealand white rabbits. Figure 18 depicts the logarithmic scale of rapamycin levels present in the vitreous at 5, 14 and 30 days after injection. Figure 19 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 84.55, 11.23, and 66.35 ng/ml at 5, 14, and 30 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 1.09, 0.19, and 1.02 ng/mg 5, 14, and 30 days after subconjunctival injection, respectively.

Example 33 - Subconjunctival injection of a suspension containing rapamycin

90 μl of the solution described in Example 30 was injected as three 30 μl doses into three sites between the sclera and conjunctiva of New Zealand white rabbits. Figure 18 depicts the logarithmic scale of rapamycin levels present in the vitreous at 5, 14 and 30 days after injection. Figure 19 depicts the logarithmic scale of rapamycin levels in the retina and choroid at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. The vitreous samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered into the vitreous. The average level of rapamycin in the vitreous was approximately 29.95, 15.30, and 49.20 ng/ml at 5, 14, and 30 days after subconjunctival injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid samples did not include the site of administration; therefore, this measurement indicated the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.55, 1.31, and 5.74 ng/mg 5, 14, and 30 days after subconjunctival injection, respectively.

Example 34 Preparation and Characterization of Rapamycin-Containing Solutions.

10.3 mg of rapamycin was dissolved in ethanol, 4995.8 mg of PEG 400 was added, and the mixture was vortexed to give a solution having a final concentration by weight of 0.205% rapamycin, 0.544% ethanol, and 99.251% PEG 400. This solution is listed as formulation #140 in Table 1. A 10 μl volume of this solution formed a non-dispersed clump when placed in the vitreous of a rabbit eye.

Example 35 - Intravitreal injection of a solution containing rapamycin

Ten μl of the solution described in Example 34 was injected into the vitreous of New Zealand white rabbit eyes. The injected solution formed a non-dispersed mass relative to the surrounding medium. Figure 20 depicts logarithmic rapamycin levels in the retina and choroid at 5 and 30 days post-injection. Figure 21 depicts logarithmic rapamycin levels in the vitreous at the same time points. For comparison, Figures 20 and 21 also depict the results of other studies described in Examples 37 and 39 below.

The vitreous was homogenized and analyzed as described in Example 2. Five rabbit eyes were analyzed at each time point. The vitreous samples may include the site of administration. The mean level of rapamycin in the vitreous was approximately 12.02 and 0.92 ng/ml at 5 and 30 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 0.08 and 0.02 ng/mg 5 and 30 days after intravitreal injection, respectively.

Example 36 Preparation and Characterization of Rapamycin-Containing Solutions

31.5 mg of rapamycin was dissolved in ethanol, 4918.9 mg of PEG 400 was added, and the solution was vortexed. The final concentrations by weight were 0.6238% rapamycin, 1.337% ethanol, and 98.035% PEG 400. This formulation was stored at 4°C until use. This solution is listed as formulation #142 in Table 1. A 10 μl volume of this solution formed non-dispersed clumps when placed into the vitreous of a rabbit eye.

Example 37 - Intravitreal injection of a solution containing rapamycin

10 μl of the solution described in Example 36 was injected into the vitreous of New Zealand white rabbit eyes. The injected solution formed a non-dispersed mass relative to the surrounding medium. FIG20 depicts logarithmic-scale rapamycin levels in the retina and choroid 5 and 30 days after injection. FIG21 depicts logarithmic-scale rapamycin levels in the vitreous at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Five rabbit eyes were analyzed at each time point. The vitreous samples may include the site of administration. The mean level of rapamycin in the vitreous was approximately 87.46 and 44.34 ng/ml at 5 and 30 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 1.40 and 0.01 ng/mg, respectively, 5 and 30 days after intravitreal injection.

Example 38 Preparation and Characterization of Rapamycin-Containing Solutions

103.5 mg of rapamycin was placed in ethanol, 4720.8 mg of PEG 400 was added, and the mixture was vortexed to give a solution having a final concentration by weight of 2.057% rapamycin, 4.116% ethanol, and 93.827% PEG 400. This solution is listed as formulation #144 in Table 1. A 10 μl volume of this solution formed non-dispersed clumps in the vitreous of rabbit eyes.

Example 39 - Intravitreal injection of a solution containing rapamycin

Ten microliters of the solution described in Example 38 were injected into the vitreous of New Zealand white rabbits. The injected solution formed a non-dispersed mass relative to the surrounding medium. FIG20 depicts logarithmic rapamycin levels in the retina and choroid at 5, 30, and 90 days after injection. FIG21 depicts logarithmic rapamycin levels in the vitreous at the same time points.

The vitreous was homogenized and analyzed as described in Example 2. Four rabbit eyes were analyzed at each time point. Vitreous samples may include the site of administration. Mean levels of rapamycin in the vitreous were approximately 120,500, 55,160, and 0.55 ng/ml at 5, 30, and 90 days after intravitreal injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous, except that the five rabbit eyes were analyzed at the 5-day and 30-day time points. The retina and choroid samples did not include the site of administration, so this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 4.75, 0.17, and 0.01 ng/mg at 5, 30, and 90 days after intravitreal injection, respectively.

Example 40 - Subconjunctival injection of a solution containing rapamycin

40 μl of the solution described in Example 17 was injected between the sclera and conjunctiva of New Zealand white rabbits. FIG22 depicts the logarithmic scale of rapamycin levels in the aqueous humor (ng/ml) at 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days after injection, and the levels of rapamycin in the cornea (ng/mg) and the retina (ng/mg) at 4, 14, 21, and 35 days after injection. The retina (R/Chrooid) levels are labeled "R/Chrooid" in FIG22.

The aqueous humor was homogenized and then analyzed by liquid chromatography and mass spectrometry. Four rabbit eyes were analyzed at each time point. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor was approximately 0.875, 1.0, 7.0, 0.725, 0.5, 0.525, 0.0, 0.125, 0.014, and 0.0485 ng/ml at 1, 4, 7, 11, 14, 21, 28, 35, 54, and 56 days after injection, respectively.

The corneas were homogenized and then analyzed by liquid chromatography and mass spectrometry. The cornea does not include the injection site, so this measurement indicates the level of rapamycin delivered to the cornea. Four rabbit eyes were analyzed at each time point. The average level of rapamycin in the cornea was approximately 0.3225, 0.1, 0.0275, and 0.0125 ng/mg 4, 14, 21, and 35 days after injection, respectively.

The retina and choroid were homogenized and analyzed as described in Example 2, and samples were taken as described above for the vitreous. The retina and choroid do not include the site of administration, therefore, this measurement indicates the level of rapamycin delivered to the retina and choroid. The average level of rapamycin in the retina and choroid was approximately 11.61, 0.2, 0.0275, and 2.655 ng/mg at 4, 14, 21, and 35 days after injection, respectively.

Example 41 - Intravitreal injection of a solution containing rapamycin

1.0 μl of the solution described in Example 17 was injected into the vitreous of New Zealand white rabbits. The injected solution formed a non-dispersed mass relative to the surrounding medium. Table 2 reports the average level of rapamycin in the aqueous humor one day after injection. For comparison, Table 2 also reports the results of the studies described in Examples 42-45 below.

The aqueous humor was homogenized and analyzed as described in Example 40. Two rabbit eyes were analyzed. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor one day after injection was approximately 0.438 ng/ml, with a standard deviation of approximately 0.141 ng/ml.

Example 42 - Intravitreal injection of a solution containing rapamycin

3.0 μl of the solution described in Example 17 was injected into the vitreous of New Zealand white rabbits. The injected solution formed a non-dispersed mass relative to the surrounding medium. Table 2 reports the average level of rapamycin in the aqueous humor one day after injection.

The aqueous humor was homogenized and analyzed as described in Example 40. Two rabbit eyes were analyzed. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor one day after injection was approximately 0.355 ng/ml, with a standard deviation of approximately 0.234 mg/ml.

Example 43 - Subconjunctival injection of a solution containing rapamycin

3.0 μl of the solution described in Example 17 was injected between the sclera and conjunctiva of New Zealand white rabbits. The injected solution formed a non-dispersed mass relative to the surrounding medium. Table 2 reports the average level of rapamycin in the aqueous humor one day after injection.

The aqueous humor was homogenized and analyzed as described in Example 40. Two rabbit eyes were analyzed. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor one day after injection was approximately 0.338 ng/ml, with a standard deviation of approximately 0.122 ng/ml.

Example 44 - Intracameral Administration of a Rapamycin-Containing Solution

5.0 μl of the solution described in Example 17 was injected into the anterior chamber of the eye of New Zealand white rabbits by injection into the anterior end of the eye. Aqueous humor was withdrawn using a syringe. Table 2 reports the average level of rapamycin in the aqueous humor 14 days after injection.

The aqueous humor was homogenized and analyzed as described in Example 40. Two rabbit eyes were analyzed. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor 14 days after injection was approximately 0.166 ng/ml, with a standard deviation of approximately 0.183 ng/ml.

Example 45 - Intracameral Administration of a Rapamycin-Containing Solution

10 μl of the solution described in Example 17 were injected into the anterior chamber of the eyes of New Zealand white rabbits. Table 2 reports the average levels of rapamycin in the aqueous humor 14 days after injection.

The aqueous humor was homogenized and analyzed as described in Example 40. Two rabbit eyes were analyzed. The aqueous humor does not include the injection site, so this measurement indicates the level of rapamycin delivered into the aqueous humor. The average level of rapamycin in the aqueous humor 14 days after injection was approximately 0.004 ng/ml, with a standard deviation of approximately 0.006 ng/ml.

All references cited herein (including patents, patent applications, and publications) are hereby incorporated by reference in their entirety, whether previously specifically cited or not.

Table 1 Liquid preparations

Table 2 Aqueous chamber rapamycin concentration

Claims (1)

1. A liquid formulation comprising 2% rapamycin by weight, 90 to 98% polyethylene glycol (PEG) by weight, and ethanol, wherein the PEG is PEG400, and wherein the liquid formulation: (i) When injected into the vitreous humor of the subject's eye, it forms a non-dispersed clump; and (ii) Not such a liquid formulation comprising 2% rapamycin, 94% PEG400, and 4% ethanol by weight of total weight.