JP2009132738A - Composition and method for administering tubulin binding agent for treatment of ocular disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of administering vascular targeting agents (particularly a tubulin binding agent) for treating malignant or nonmalignant vascular proliferative diseases in ocular tissues. <P>SOLUTION: The invention relates to administration of vascular targeting agents, particularly the tubulin binding agent, for treating ocular neovascularization, ocular tumors, and conditions such as diabetic retinopathy, retinopathy of premature baby, retinoblastoma and macular degeneration. The method provided for treating ocular diseases involves the following processes: (a) a process of preparing medication including a pharmacologically effective dosage of the tubulin binding agent and (b) a process of administering the pharmacologically effective dosage to a subject requiring the treatment. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the administration of vascular targeting agents (particularly tubulin binding agents) for the treatment of eye diseases.

  The eye is basically one of the most important organs in life. The ability to maintain eye health is all important due to aging, diseases and other factors that can adversely affect vision. The primary cause of blindness is the inability to introduce drugs or therapeutic factors into the eye and the inability to maintain these drugs or factors at therapeutically effective concentrations. Oral inoculation of the drug or infusion of the drug at a site other than the eye provides the drug systemically. However, such systemic administration does not provide an effective level of that drug specifically for the eye, and therefore often administers unacceptably high levels of factors to achieve effective intraocular concentrations. May be required.

  A plaque is a retinal region containing elevated concentrations of photosensory cells responsible for fine and detailed vision (a general anatomical view of the human eye is shown in FIG. 1). Macular degeneration is an ambiguous historical name given to a poorly understood group of diseases that cause macula photosensory cells to lose function. The result of macular degeneration is significant central vision and loss of detailed vision. Patients suffering from macular degeneration experience a blank spot in the center of their field of view and often lose the ability to read small prints (Source: Macro Generation Foundation, San Jose, CA: www.eyesight.org).

  More than 12 million Americans have some form of macular degeneration. One in six Americans between the ages of 55 and 64 suffer from macular degeneration, and the incidence of the disease increases with age. Of the estimated 12 million patients with macular degeneration, 1.2 million each year suffer from severe central vision loss. Each year 200,000 individuals lose all of their central vision in one or both eyes.

  The exact cause of macular degeneration is unknown, but the structure of the macula provides clues as to how the disease can be initiated. Plaques contain highly active photoreceptors that consume large amounts of energy. Producing abundant oxygen and nutrients is required to generate this energy. The plaque has one blood flow with the highest velocity through its supply vessel (also known as the choroid). Anything that interferes with this rich vascular supply can cause plaque dysfunction. Oxygen-depleted plaques respond by generating cytokines that signal endothelial cell proliferation and neovascularization.

  There are two basic types of macular degeneration (dry and wet forms). About 85% to 90% of the causes of macular degeneration are dry. In the dry form of the disease, retinal deterioration is associated with the formation of yellow deposits under the plaques known as crystal cavities. Crystallographic deposits correlate with a decrease in the thickness of retinal cells, including plaques. The amount of central vision loss is directly related to the location and severity of retinal thickening induced in the crystal cavity. The dry form of macular degeneration tends to progress more slowly than the wet form of the disease. There is no effective treatment for dry form macular degeneration. A small number of individuals suffering from dry forms of macular degeneration progress to wet forms of macular degeneration. FIG. 2 shows normal and dry forms of macular degeneration.

  Wet form of macular degeneration is a rapidly progressing disease that almost always results in severe vision loss. Visual loss associated with wet macular degeneration is a result of subretinal neovascularization. Rapid proliferation of the subretinal blood vessels causes the retinal cell layer above it to bend and detach from the nutrient-rich choroid. In extreme cases of wet macular degeneration, the vessels infiltrate the retina and proliferate into the vitreous humor during growth. There are several treatments for wet forms of neovascularization, all of which are far from satisfactory. FIG. 3 shows normal retina and wet macular degeneration.

  The current standard treatment for macular degeneration is laser photocoagulation. An ophthalmologist performing laser photocoagulation positions abnormal vessels using fluorescent angiography and selectively burns the vessels using laser ablation techniques. The side effect of laser surgery is the destruction of the retinal layer that lies just above the abnormal vessels. Patients treated with laser photocoagulation have measurable loss of vision immediately after treatment, which is an unacceptable negative side effect. In general, laser surgery is considered an ad hoc treatment that is only moderately effective in slowing down the disease.

Photodynamic therapy is the current level of treatment technology for macular degeneration. The US Food and Drug Administration has approved verteporfin for injection (Visudyne ^™ developed by Ciba Vision & QLT) for treating wet age-related macular degeneration. Patients being treated with photodynamic therapy are injected with a photoreactive compound (verteporfin) and immediately treated with a non-destructive ophthalmic laser. The ophthalmologist performing the surgery identifies the abnormal vessel and directs the laser beam to the abnormal vessel. Verteporfin, when activated by a laser, produces a transient energy burst that effectively burns all cells in the vicinity of the activated molecule (Source: HHS News, US Department of Health and Human Services, 2000). April 13,

  Ionizing radiation is used to kill growing vessels (proliferating cells are more sensitive to radioactivity than quiescent cells). Ionizing radiation is usually administered in a beam that is large enough to expose the majority of the eye. In 1993, the University of Belfast in North Ireland group reported that they tried X-rays for a small number of patients suffering from wet macular degeneration. The positive result is supported by several similar studies using X-rays conducted by other European research teams.

  Another debilitating eye disease is retinopathy of prematurity (ROP). ROP is an eye disease that occurs in a significant proportion of premature infants. The last 12 weeks of full-term delivery (28-40 weeks) is a particularly active month in fetal eye development. Prenatal development of the retinal blood supply (choroid) begins in the optic nerve at 16 weeks and progresses towards birth (40 weeks) towards the anterior region of the retina in a radial fashion. When childbirth is immature, the retinal vasculature does not have enough time to occur and the anterior edge of the retina becomes oxygen deficient. Lack of anterior retinal oxygenation is a potential cause of ROP (Source: The Association of Retinopathy of Prerequisite and Related Diseases, Franklin, MI).

  In premature babies, a significant portion of the anterior retina is deficient in adequate blood supply. The oxygen-deficient anterior retina responds by signaling for neovascular growth. Abnormal neovascularization in the zone between the anterior and retinal retinas initiates a cascade of events with severe pathological consequences. As the neovasculature grows in response to a chemical signal, an arteriovenous shunt is formed in the zone between the vascularized posterior retina and the anterior retina where there are no vessels. These vascular shunts gradually expand, become thicker and rise more. The neovasculature is achieved by infiltrating fibroblasts, which produce fibrotic scar tissue. Eventually, a scar tissue ring attached to the retina and vitreous gel is formed. This scar tissue ring may expand 360 ° around the inside of the eye. When the scar tissue comes into contact, the scar tissue pulls the retina and causes retinal detachment. If sufficient scar tissue is formed, the retina can be completely detached. Premature infants are at risk of developing ROP. Because premature babies have been taken out of the protective environment of the uterus and exposed to various angiogenic stimuli, including medications, high levels of oxygen, and light and temperature fluctuations. is there. Some or all of these factors may have an effect on the development of ROP. Fortunately, most premature babies do not develop ROP, and most infants with ROP improve naturally. When ROP develops, ROP usually occurs between 34 and 40 weeks after conception, regardless of the gestational age at birth.

  A technique called cryotherapy has been shown to have a beneficial effect for the treatment of ROP. Cryotherapy involves placing a sub-freezing probe on the outer wall (sclera) of the eye. This probe produces a zone of ice crystallization on the retina surface between the sclera and the vitreous. Multiple applications of cryotherapy are performed to treat the entire non-vascular area (which is in front of the neovascular edge). The treatment of the rim itself is avoided. Because this edge tends to bleed and, when frozen, causes vitreous hemorrhage.

  The mechanism of action of cryotherapy is not fully understood. A practical hypothesis is that cryotherapy probably damages the anterior retinal layer without blood vessels. This damage results in thickening of the retina that can facilitate the diffusion of oxygen into the remaining living cells. Furthermore, the cold treated retina has fewer live cells and thus has a reduced demand for oxygen. Reduced oxygen demand reduces angiogenic stimulation and stops neovascularization. Cryotherapy was found to reduce the risk of retinal detachment from 43% of untreated eyes to 21% of treated eyes. However, cryotherapy has potential complications; this procedure is often performed under general anesthesia, which can be dangerous for premature infants.

  Laser photocoagulation as described herein above uses a similar principle in the treatment of ROP. This laser treatment is applied to the anterior retina that does not yet have a blood supply. The purpose of this procedure is to eliminate the abnormal vessel before placing it below enough scar tissue to cause retinal detachment. In addition, the anterior retinal area without blood vessels is thinned by the laser, oxygen demand is reduced, and angiogenic stimulation is reduced, much like cold therapy. Laser therapy is superior to cryotherapy in that it is directed to the retina but not to the entire thickness of the eye wall. Since laser therapy is painless with fewer tissues, inflammation after treatment is greatly reduced. Laser therapy is superior when compared to cryotherapy. This is because the need for anesthesia is reduced.

  If laser therapy or cryotherapy is unsuccessful in stopping the progression of ROP, several surgical procedures are available. If there is a shallow retinal detachment due to small traction from the fibro-vascular scar tissue, a procedure called scleral fold may be beneficial. Scleral folds include placing a silicon band around the eyeball equator and tightening the silicon band to create a slight dent inside the eye. This band reduces vitreal gel traction and pulls on fibrous scar tissue and the retina. This allows the retina to flatten on the eye wall and restore normal function. Infants with scleral folds can maintain good visual acuity in the eye, especially when the plaques do not detach. This surrounding band usually needs to be removed after months or years. This is because the eye continues to grow, resulting in gradually increasing eye compression and induced myopia.

  In late stage ROP, the retina completely detaches due to scar tissue on the retina, and scleral folding is insufficient to reduce traction. For these infants, vitrectomy can be considered. Vitreous resection involves making several small incisions in the eye and removing the vitreous gel using a suction / cutting device. The vitreous is replaced with saline and the eye morphology is maintained. The eye can maintain its shape and pressure indefinitely without vitreous gel. After the vitreous is removed, scar tissue on the retina can be peeled off or excised, thereby causing the retina to relax and become more squeezed against the eye wall. It can take several weeks for the retina to reattach after surgery. During treatment, the retina usually does not reattach when tears occur in the retinal hole and in the retina. Although the eye lens often must be removed to allow complete dissection of the scar tissue, several new techniques that can preserve the lens have been tried.

  However, the success rate of vitrectomy for ROP is limited. Published anatomical success rates (meaning obtaining retinas that reattach to the eye wall) range from 25% to 50% of patients undergoing surgery. The functional success rate (which means ability to look good) is significantly lower. Of the eyes that have "successful" vitrectomy surgery (anatomical success), only about 1/4 are good enough to reach and grasp the object, or enough to recognize the pattern Can see.

  Another debilitating eye disease occurs in patients with diabetes mellitus. About 14 million Americans have diabetes mellitus. In addition to causing numerous systemic complications (eg, renal failure, hypertension, and cardiovascular disease), diabetes is one of the leading causes of blindness among working-age Americans. In fact, the risk of blindness for patients with diabetes is 25 times higher than that of the general population. Many patients with diabetic eye problems are asymptomatic despite the presence of diseases that threaten vision. If the diabetic eye disease is left untreated, the disease can result in severe vision loss. Vision loss due to diabetes can be caused by several mechanisms, and treatment needs to be adjusted to the needs of the individual. (Source: The Center for Disease Control, “The Prevention and Treatment of Diabetes Mellitus-A Guide for Primary Care Practitioners”): www.cdc.

  Many diabetics notice blurred vision when their blood sugar is particularly high or particularly low. This blurred vision results from a change in the shape of the lens of the eye and usually reverses when the blood glucose returns to normal. Diabetes is a disease that not only affects a patient's blood glucose level but also affects blood vessels. Symptoms related to diabetes (including increased blood pressure) cause damage to the microcirculatory system including capillaries related to the retina. Capillary damage results in a decrease in blood flow to isolated areas of the retina. In addition, damaged blood vessels tend to leak, which causes swelling in the retina.

  There are two main categories of diabetic eye diseases. The first category is called background diabetic retinopathy or nonproliferative retinopathy. This is essentially the earliest stage of diabetic retinopathy. This stage is characterized by damage to small retinal blood vessels, which results in the leaching of fluid (blood) into the retina. Most vision loss during this phase is due to this fluid accumulation in the plaque. This fluid accumulation is called macular edema. This fluid accumulation can cause temporary visual loss or permanent visual loss. The second category of diabetic retinopathy is called proliferative diabetic retinopathy. Proliferative retinopathy is the end result of diabetes-induced damage maintained by the retinal capillary bed (choroid). Damage to the choroid causes oxygen depletion in the retina. Retinal tissue responds to its hypoxic environment by producing angiogenic cytokines that stimulate neovascularization. As previously described, retinal neovascularization causes bleeding in the eye, retinal scar tissue, retinal detachment, and any one of these symptoms can cause vision loss or blindness. Diabetic patients also often suffer from neovascular glaucoma, which indicates rubeosis, a blood vessel that grows on the iris that causes angle closure.

  Diabetic retinopathy can occur in both type I diabetic patients (onset of diabetes before age 40) and type II diabetics (onset after age 40), whereas diabetic retinopathy is observed in type I patients. It tends to be more general and more severe. Type II diabetic patients are often not diagnosed until the patient has perennial diabetes, but diabetic retinopathy may be present in type II patients at the time diabetes is discovered.

  Treatment of diabetic retinopathy depends on a number of factors, including the type and extent of retinopathy, the relevant ocular factors (eg, cataract or vitreous hemorrhage), and the patient's medical history. Treatment options include the same options discussed for ROP (ie, laser photocoagulation, cryotherapy (freezing), and vitrectomy surgery). Times due to diabetic retinopathy are preventable in most cases.

  Any type of intraocular cancerous tumor is almost uncommon. However, ocular tumors are very serious in that uveal (eye) cancer generally metastasizes to and from other body regions. Uveal melanoma, the most common major malignancy of the eye, occurs in 7 out of 1 million people in the general population per year (less than 1/10 of the incidence of lung cancer). Retinoblastoma occurs as a childhood disease with the same frequency as hemophilia. These two intraocular tumors are very different and are related only by anatomical proximity. Treatment options for ocular cancer depend on where the cancer is in the eye, how far the cancer has spread, and the patient's general health and age (source: The Eye Cancer Network). : Www.eyecancer.com; OncoLink: cancer.med.upenn.edu).

  Retinoblastoma is a cancer of one or both eyes that occurs in young children. There are approximately 350 newly diagnosed cases per year in the United States. Retinoblastoma affects 1 in 15,000-30,000 surviving infants born in the United States. Retinoblastoma affects children of all races as well as both boys and girls.

  Retinoblastoma originates in the retina, the light-sensitive layer of the eye that makes things visible to the eye. Treatment of retinoblastoma is individualized for each patient and depends on the child's age, involvement in one or both eyes, and whether the cancer has spread to other body parts . If left untreated, the child dies. Treatment for retinoblastoma includes excision, external beam irradiation, radioactive plaque, laser therapy, cryotherapy, and chemoduction.

  Removal is the most common form of treatment for retinoblastoma. During the removal, the eye is surgically removed. This removal is necessary because complete removal of the cancer is the only way. It is impossible to remove cancer from within the eye without removing the entire eye. Partial excision is possible for some other ocular cancers, but it is dangerous and may still contribute to cancer transmission for retinoblastoma patients.

  Where both eyes are involved, sometimes the more involved, or “bad” eye, is removed while the other eye is a visual preservation procedure (eg, external, described below) Can be treated with one of beam irradiation, plaque therapy, cryotherapy, laser treatment, and chemoduction).

  External beam irradiation has been used as a method for preserving eyes and vision since the early 1900s. Retinoblastoma is sensitive to radiation and often this treatment is successful. This irradiation treatment is performed on an outpatient basis 5 times per week for a length of 3-4 weeks. Custom gypsum molds are made to prevent movement of the head during treatment, and sometimes sedatives are prescribed before treatment.

  Tumors usually appear smaller (regressed) and scarred after external beam irradiation treatment, but rarely disappear completely. In fact, tumors can even become clearer as they shrink. Because the pinkish gray tumor mass is replaced with white calcium. Immediately after the procedure, the skin can be burned or a small hair can be lost behind the head from the exit location of the beam. Long term effects after external beam irradiation include cataracts, radiation retinopathy (blindness and retinal exudates), visual loss, and temporary bone suppression (bones on the side of the head that do not grow normally). Irradiation can also increase the child's risk of developing other tumors outside the eye for children who have aberrant genes in any cell of the body.

  A radioactive plaque is a disc of radioactive material developed in the 1930s to irradiate retinoblastoma. Today, the isotope iodine 125 is used and this plaque is made for each child. The child must usually be hospitalized for this surgery and undergo two separate surgeries (one to insert and one to remove plaque) for 3-7 days. There must be.

  Laser therapy, sometimes referred to as photocoagulation or laser hyperthermia (these are two different techniques), is a non-invasive procedure for retinoblastoma. The laser effectively destroys relatively small retinoblastomas. This type of treatment is usually performed by focusing light through the pupil over and around the cancer in the eye. In recent years, a novel delivery system of lasers (referred to as diopexy probes) has made it possible to treat the cancer by casting light through the eye wall rather than through the pupil. Laser treatment is performed under local or general anesthesia and usually does not have any post-operative pain associated with the treatment and does not require any post-operative medication. The laser can be used alone or in addition to external beam irradiation, plaque, or cryotherapy.

  Cryotherapy can also be performed on patients suffering from retinoblastoma. Cryotherapy is performed under local or general anesthesia to freeze smaller retinoblastoma tumors. A pen-like probe is placed in the sclera adjacent to the tumor and the tumor is frozen. Cryotherapy usually has to be repeated a number of times in order to substantially destroy all of the cancer cells. An adverse side effect of cryotherapy is that the eyelids and eyes swell for 1 to 5 days; sometimes this swelling is so severe that children can open their eyes (open their eyes) for several days Can not. Eye drops or ointments are often given to reduce swelling.

  Systemic chemotherapy is the treatment of retinoblastoma using chemotherapy. Chemotherapy is generally administered intravenously to children and through the bloodstream results in tumor shrinkage within a few weeks if successful. Chemotherapy using one or more drugs can be performed once, twice or more. Depending on the drug and the institution, the child may or may not be hospitalized during this process. After chemotherapy, the child is reexamined and the remaining tumor is treated using cryotherapy, laser, or radioactive plaque. Children may need to be treated as many as 20 times every 3 weeks, under anesthesia, retesting the eye.

  Although rare, if retinoblastoma is treated immediately, retinoblastoma can spread (metastasize) from outside the eye to the brain, central nervous system (brain and spinal cord), and bone. In this case, chemotherapy is prescribed by pediatric oncologists and is administered through peripheral blood vessels or into the brain for months to years after the initial diagnosis of metastatic disease.

  Tumors other than retinoblastoma and melanoma occur in the eye, and these are often precursors of disease elsewhere. Choroidal metastases are most likely to cause intraocular malignancy and may be the first sign of systemic malignancy. Choroidal metastasis is similar to nonpigmented melanoma. They have an appearance similar to melanoma on fluorescent fundus angiograms and show slight echogram differences on ultrasound recordings. However, choroidal metastasis is more likely to grow more rapidly and cause large exudative retinal detachment.

  In general, the prognosis for survival is poor once metastatic disease is found in the eye. However, as survival in patients with systemic cancer improves, successful treatment of eye metastases plays an increasingly important role in maintaining good quality of life.

  Primary ocular lymphomatosis is one of the most intriguing intraocular tumors. The relationship between the primary CNS lymphoma and its tendency to grow in the subretinal pigment epithelial space in the absence of lymphoid tissue is two real attractive aspects of this highly aggressive lymphoma . The clinical manifestation of primary ocular lymphoma is well-known to mimic the benign uveoid entity, thus delaying accurate diagnosis for months. Neoplastic cells in ocular lymphoma can continue to be constrained in the space between the retinal pigment epithelium and Bruch's membrane. Vitritis associated with these aggregations of lymphoma often consists of reactive lymphocytes and vitreous biopsy can be non-diagnostic. This actually led to the misunderstanding that it is difficult to elucidate intraocular cytology if the surgeon was not taking tumor cells. The positive yield from an intraocular biopsy can be increased in some cases when the surgeon performs an inhalation biopsy via a retinotomy in the subretinal pigment epithelial space. Primary ocular lymphoma consists of large, cytologically atypical cells that stain positive for leukocyte common antigen. Suction is usually associated with a large amount of necrotic debris. Immunophenotypic analysis has been problematic in the past. Some early studies failed to find any surface markers and concluded that the ocular lymphoma was a non-hormone producing cell tumor. Pretreatment of cells with hyaluronidase increased the yield of immunopathological studies.

  Another form of eye cancer is choroidal melanoma. Choroidal melanoma is a primary cancer of the eye. It is not a cancer that originates from pigment cells in the choroid of the eye and has spread elsewhere and spread to the eye. Some choroidal melanomas are more life threatening than other cancers, but almost all should be treated as if they were malignant. Some choroidal melanomas appear to remain inactive and do not grow. Most spread slowly over time, causing blindness. These tumors can spread to other parts of the body, ultimately resulting in death. Many cases of ocular melanoma metastasis to the liver have been reported (source: The Eye Cancer Network: www.eyecancer.com).

  For many years, a useful treatment for choroidal melanoma has been removal. If the tumor has not spread to other parts of the body, removal of the eye generally removes the tumor completely from the patient. Since World War II, radiation treatment has been used for choroidal melanoma. Over the past 20 years, this method of treatment has been improved. Irradiation at the appropriate dose rate and the appropriate physical form is intended to eliminate proliferating tumor cells without causing damage to sufficient normal tissue that requires eye removal. As the cells die, the tumor shrinks but usually does not disappear completely. The most promising and widely available method for irradiating intermediate choroidal melanoma involves constructing small plaques with radioactive pellets adhered to one side. However, irradiation is usually accompanied by deleterious side effects such as vomiting and hair loss.

  High energy particles from the cyclotron (irradiation of helium ions or proton beams) can also be used to irradiate the tumor. First, an operation is performed to sew a small metal clip to the sclera so that the particle beam can be accurately aimed. Treatment is given over several consecutive days. The instruments required for these procedures are only available at some medical centers around the world. Although good results have been reported in some patients, many patients treated with this method followed only for a few years. Thus, the long-term results of these forms of radiation therapy compared to the more commonly used plaques are unknown.

  Over the years, other treatments have been used for a small number of patients. Photocoagulation using white light or laser light was used to burn small tumors, and cryotherapy was used to kill them by freezing the tumors. These techniques are believed to work only for very small tumors. Some doctors combined laser or cryotherapy with radiation, but such treatment was experimental. Some patients have undergone eye wall resection or related procedures to remove the tumor from their eyes. These treatments are considered experimental by most physicians and were used only for a few tumors. No treatment is available that can ensure tumor destruction, vision protection, or ensuring a normal life span.

  Another eye cancer is intraocular melanoma, in which rare cancers are found in the part of the eye called the uvea. The uvea contains cells called melanocytes that contain pigment. When these cells become cancerous, the cancer is called melanoma. The uvea includes the iris (the colored part of the eye), the ciliary body (muscle in the eye), and the choroid (the tissue layer behind the eye). The iris changes the amount of light that enters and exits the eye. The ciliary body changes the shape of the lens inside the eye so that it is in focus. The choroid layer is next to the retina and is the part of the eye that produces the image. If there is a melanoma that begins in the iris, it can appear as a black spot on the iris. If the melanoma is in the ciliary body or choroid, it may have a blurred vision or no symptoms. The cancer can then grow before it is noticed (Source: The Eye Cancer Network: www.eyecancer.com).

  The change in recovery (prognosis) from intraocular melanoma depends on the size of the cancer and the cell type of the cancer if the cancer is in the eye and if the cancer has spread. Treatment exists for all patients with intraocular melanoma. Three treatment types: surgery (removal of cancer), radiation therapy (high dose x-rays or other high energy rays to “kill” cancer cells), and photocoagulation (destroying blood vessels that feed the tumor) ) Is generally administered.

Surgery is the most common treatment for intraocular black species. The doctor can remove the cancer using one of the following procedures:
-Iris resection-removal of only part of the iris;
-Iris trabeculectomy-removal of part of the iris and supporting tissue around the cornea (clean layer covering the front of the eye);
-Iris ciliary resection-removal of the iris and part of the ciliary body;
-Choroidectomy-removal of part of the choroid;
-Removal-Removal of the entire eye.

  Radiation therapy can also be used to apply x-rays or other high energy rays to the area where the cancer cells are present in order to kill the cancer cells and shrink the tumor. Radiation can be used alone or in combination with surgery. A photocoagulation procedure can also be used, where a minimal beam of light (usually from a laser) can be applied to the eye to destroy blood vessels and kill the tumor.

  The overwhelming majority of therapies proposed for the treatment of eye diseases (especially subretinal neovascularization and eye tumors) initially utilize surgery or radiation treatment. When a patient is treated with medication alone or after surgery, drug administration is generally systemic, either by injection or oral. As noted above, both surgical or radiation treatments for eye diseases are both painful, often require a long recovery period, and can be followed by adverse side effects. In addition, systemic administration via oral ingestion of the drug or injection at a site other than the eye is effective, requiring administration of high levels of drug that are often unacceptable to achieve effective intraocular concentrations. Often provided in non-amounts. Thus, there is a great need for successful non-systemic treatment for the treatment of cancer diseases such as corneal and retinal neovascularization. Moreover, delivering drugs and medications to the eye without harmful side effects remains a major challenge. The present invention provides a therapy that provides effective non-systemic administration of tubulin binding agents for the treatment of cancer diseases with minimal side effects.

(Summary of the Invention)
The present invention is directed to the administration of vascular targeting factors (“VTA”) (particularly tubulin binding agents) for the treatment of malignant or non-malignant vascular proliferative disorders in ocular tissues.

  Neovascularization of ocular tissue is a pathogenic condition characterized by vascular proliferation and occurs in various eye diseases with varying degrees of visual dysfunction. Patients with non-malignant vascular proliferative disorders (eg, treatment of neovascularization associated with wet macular degeneration, proliferative diabetic retinopathy or premature retinopathy where most treatment options are not available) In another embodiment, the present invention provides for the administration of VTA to a pharmacological control of neovascularization associated with malignant angioproliferative disorders (eg, ocular tumors). To do.

  The blood-retinal barrier (BRB) is composed of tightly connected, specialized, non-windowed endothelial cells that form a transport barrier for specific substances between retinal capillaries and retinal tissue. The cornea and retinal neovascularization associated with retinopathy, which closely resemble the blood vessels associated with solid tumors, are abnormal. Tubulin-binding drugs, inhibitors of tubulin polymerization, and vascular targeting factors can attack abnormal blood vessels. This is because these blood vessels do not share structural similarity with the vascular retinal barrier. Tubulin-binding drugs can stop the progression of diseases that mimic diseases in which blood vessels share structural similarities with tumor-vasculature. Local (non-systemic) delivery of tubulin binding agents to the eye can be achieved using intravitreal injection, subthononic injection, eye drop iontophoresis, and implants and / or inserts. Systemic administration is accomplished by administering a tubulin binding agent into the bloodstream at a site that is a measurable distance away from the diseased organ or diseased tissue or diseased organ or diseased tissue (in this case, the eye). obtain. Preferred modes of systemic administration include parenteral administration or oral administration.

  The details of one or more embodiments of the invention are set forth in the accompanying description below. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and the preferred methods and materials are now described. Other features, objects and advantages of the invention will depart from this description. In this specification and the appended claims, the singular forms also include the plural unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All of the patents and publications listed herein are hereby incorporated by reference.

For example, the present invention provides the following items.
(Item 1)
  A method for treating an eye disease comprising the following steps:
  a) preparing a dosage containing a pharmaceutically effective dosage of a tubulin binding agent;
  b) administering the pharmaceutically effective dosage to a subject in need of the treatment;
Including the method.
(Item 2)
  2. The method of item 1, wherein the tubulin binding agent is combretastatin A4.
(Item 3)
  2. The method of item 1, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 4)
  2. The method of item 1, wherein the ocular disease can comprise retinal neovascularization, choroidal neovascularization, and ocular tumor neovascularization.
(Item 5)
  2. The method of item 1, wherein the ocular disease can include diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 6)
  2. The method of item 1, wherein the eye disease may comprise corneal neovascularization and macular degeneration.
(Item 7)
  2. The method of item 1, wherein the pharmaceutically effective dosage is administered non-systemically to the eye of the subject.
(Item 8)
  Said pharmaceutically effective dosage is via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, eye drops, eye gels, eye ointments, iontophoresis, as well as eye implants 2. The method of item 1, wherein the method is administered non-systemically to the eye by and / or ocular insert.
(Item 9)
  8. The method of item 7, wherein the pharmaceutically effective dosage administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml. .
(Item 10)
  2. The method of item 1, wherein the pharmaceutically effective dosage is systemically administered to the subject.
(Item 11)
  The method of item 1, wherein the pharmaceutically effective dosage is administered parenterally.
(Item 12)
  The pharmaceutically effective dosage administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 11. The method of item 10, comprising an amount of combretastatin A4 prodrug in the range of.
(Item 13)
  Item 2. The method according to Item 1, wherein the subject is a mammal.
(Item 14)
  A pharmaceutical medicament for treating an eye disease, wherein a therapeutically effective amount of a tubulin binding agent to reduce ocular neovascularization is administered to a subject in need of said treatment. For pharmaceutical purposes, containing in combination with a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
(Item 15)
  Item 15. The pharmaceutical agent according to Item 14, wherein the tubulin-binding agent is combretastatin A4.
(Item 16)
  Item 15. The pharmaceutical agent of item 14, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 17)
  15. The pharmaceutical medicament of item 14, wherein the ocular disease can comprise retinal neovascularization, choroidal neovascularization, and ocular neovascularization.
(Item 18)
  Item 15. The pharmaceutical medicament of item 14, wherein the eye disease may comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 19)
    Item 15. The pharmaceutical medicament of item 14, wherein the eye disease may comprise corneal neovascularization and macular degeneration.
(Item 20)
  15. A pharmaceutical medicament according to item 14, wherein the pharmaceutically effective dosage is administered non-systemically to the eye of the subject.
(Item 21)
  Said pharmaceutically effective dosage is via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by iontophoresis, and by ocular implants and / or ocular inserts, Item 15. The pharmaceutical medicament according to Item 14, which is administered non-systemically to the eye.
(Item 22)
  21. The pharmaceutical of item 20, wherein the pharmaceutically effective dosage administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml. Medicine.
(Item 23)
  Item 15. The pharmaceutical agent of item 14, wherein the pharmaceutically effective dosage is systemically administered to the subject.
(Item 24)
  Item 15. The pharmaceutical medicament according to Item 14, wherein the pharmaceutically effective dosage is administered parenterally.
(Item 25)
  The pharmaceutically effective dosage administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 24. The pharmaceutical medicament of item 23, comprising an amount of combretastatin A4 prodrug in the range of.
(Item 26)
  Item 15. The pharmaceutical medicament according to Item 14, wherein the subject is a mammal.
(Item 27)
  A method of treating or preventing ocular neovascularization in a subject, the method comprising administering to the subject a pharmaceutically effective dosage of a tubulin binding agent.
(Item 28)
  28. The method of item 27, wherein the tubulin binding agent is combretastatin A4.
(Item 29)
  28. The method of item 27, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 30)
  28. The method of item 27, wherein the ocular neovascularization may comprise retinal neovascularization, choroidal neovascularization, and ocular tumor neovascularization.
(Item 31)
  28. The method of item 27, wherein the pharmaceutically effective dosage is administered non-systemically to the eye of the subject.
(Item 32)
  Said pharmaceutically effective dosage is via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by iontophoresis, and by ocular implants and / or ocular inserts, 28. The method of item 27, wherein the method is administered non-systemically to the eye.
(Item 33)
  32. The method of item 31, wherein the pharmaceutically effective dosage administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml. .
(Item 34)
  28. The method of item 27, wherein the pharmaceutically effective dosage is systemically administered to the subject.
(Item 35)
  32. The method of item 30, wherein the pharmaceutically effective dosage is administered parenterally.
(Item 36)
  The pharmaceutically effective dosage administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 35. The method of item 34, comprising an amount of combretastatin A4 prodrug in the range of.
(Item 37)
  31. A method according to item 30, wherein the subject is a mammal.
(Item 38)
  A method of treating or preventing an ocular tumor, comprising administering a pharmaceutically effective dosage of a tubulin binding agent to a subject in need of said treatment or prevention. .
(Item 39)
  40. The method of item 38, wherein the tubulin binding agent is combretastatin A4.
(Item 40)
  40. The method of item 38, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 41)
  40. The method of item 38, wherein the ocular tumor can comprise retinoblastoma, primary ocular lymphoma, choroidal melanoma, and intraocular melanoma.
(Item 42)
  40. The method of item 38, wherein the pharmaceutically effective dosage is administered non-systemically to the eye of the subject.
(Item 43)
  Said pharmaceutically effective dosage is via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, eye drops, eye gels, eye ointments, iontophoresis, as well as eye implants 39. The method of item 38, wherein the method is administered non-systemically to the eye and / or by an ocular insert.
(Item 44)
  45. The method of item 42, wherein the pharmaceutically effective dosage administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml. .
(Item 45)
  40. The method of item 38, wherein the pharmaceutically effective dosage is administered systemically to the subject.
(Item 46)
  40. The method of item 38, wherein the pharmaceutically effective dosage is administered parenterally.
(Item 47)
  The pharmaceutically effective dosage administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 46. The method of item 45, comprising an amount of combretastatin A4 prodrug in the range of.
(Item 48)
  40. The method of item 38, wherein the subject is a mammal.
(Item 49)
  A pharmaceutical composition comprising a therapeutically effective amount of a tubulin binding agent for administration to a subject suffering from an eye disease.
(Item 50)
  50. The pharmaceutical composition of item 49, wherein the tubulin binding agent is combretastatin A4.
(Item 51)
  50. The pharmaceutical composition of item 49, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 52)
  50. The pharmaceutical composition of item 49, wherein the ocular disease can comprise retinal neovascularization, choroidal neovascularization, and ocular tumor.
(Item 53)
  50. The pharmaceutical medicament of item 49, wherein the eye disease can comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 54)
    50. The pharmaceutical medicament of item 49, wherein the ocular disease can comprise corneal neovascularization and macular degeneration.
(Item 55)
  50. The pharmaceutical composition of item 49, wherein the therapeutically effective amount of a tubulin binding agent is administered non-systemically to the eye of the subject.
(Item 56)
  The composition may be administered via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by eye gel, by eye ointment, by iontophoresis, and by ocular implants and / or ocular insertions. 50. The pharmaceutical composition of item 49, wherein the pharmaceutical composition is administered non-systemically to the eye of the subject.
(Item 57)
  56. The pharmaceutical composition of item 55, wherein the therapeutically effective amount administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml.
(Item 58)
  50. The pharmaceutical composition of item 49, wherein said therapeutically effective amount of combretastatin A4 prodrug is systemically administered to said subject.
(Item 59)
  50. The pharmaceutical composition of item 49, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered parenterally.
(Item 60)
  The therapeutically effective amount administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 59. The pharmaceutical composition of item 58, containing an amount of combretastatin A4 prodrug in the range of.
(Item 61)
  50. The pharmaceutical composition of item 49, wherein the subject is a mammal.
(Item 62)
  A pharmaceutical dosage form comprising a therapeutically effective amount of a combretastatin A4 prodrug and a pharmaceutically acceptable carrier or excipient, the pharmaceutical dosage form administering the dosage form to a subject A pharmaceutical dosage form for treating an ophthalmic disease in said subject.
(Item 63)
  63. The pharmaceutical dosage form of item 62, wherein the ocular disease can include retinal neovascularization, choroidal neovascularization, and ocular tumor.
(Item 64)
  63. The pharmaceutical dosage form of item 62, wherein the eye disease can comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 65)
    63. The pharmaceutical dosage form of item 62, wherein the ocular disease can comprise corneal neovascularization and macular degeneration.
(Item 66)
  63. The pharmaceutical dosage form of item 62, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered non-systemically to the eye of said subject.
(Item 67)
  The composition may be administered via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by eye gel, by eye ointment, by iontophoresis, and by ocular implants and / or ocular insertions. 63. The pharmaceutical dosage form according to item 62, wherein the pharmaceutical dosage form is administered non-systemically to the eye of the subject by product.
(Item 68)
  70. The pharmaceutical dosage form of item 66, wherein said therapeutically effective amount administered non-systemically comprises combretastatin A4 prodrug in an amount ranging from about 0.1 mg / ml to about 100 mg / ml.
(Item 69)
  63. The pharmaceutical dosage form of item 62, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered systemically to said subject.
(Item 70)
  64. The pharmaceutical dosage form of item 62, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered parenterally.
(Item 71)
  The therapeutically effective amount administered systemically is about 0.1 mg / m ² ~ About 120mg / m ² 70. The pharmaceutical dosage form according to item 69 comprising an amount of combretastatin A4 prodrug in the range of.
(Item 72)
  65. A pharmaceutical dosage form according to item 64, wherein the subject is a mammal.
(Item 73)
  A method of treating an ophthalmic disease comprising: tubulin binding to an eye of a subject in need of treatment, in the eye, ranging between about 1 nM to about 100 mM of aqueous humor tissue. A method by administering a tubulin binding agent at a dosage sufficient to achieve a concentration of the agent.
(Item 74)
  74. The method of item 73, wherein the ocular disease can include retinal neovascularization, choroidal neovascularization, and ocular tumor neovascularization.
(Item 75)
  74. The method of item 73, wherein the eye disease can comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 76)
  74. The method of item 73, wherein the ocular disease can include corneal neovascularization and macular degeneration.
(Item 77)
  74. The method of item 73, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 78)
  Said tubulin binding agent is injected via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by eye gel, by eye ointment, by iontophoresis, and / or ocular implants and / or 74. The method of item 73, wherein the method is administered to the subject's eye by an eye insert.
(Item 79)
  A pharmaceutical composition for topical administration to the eye of a subject suffering from an eye disease, said pharmaceutical composition being pharmaceutically acceptable for administration to a subject in need thereof A pharmaceutical composition comprising a tubulin binding agent in combination with a suitable carrier, excipient, diluent, or adjuvant.
(Item 80)
  80. The pharmaceutical composition of item 79, wherein the tubulin binding agent is a combretastatin A4 prodrug.
(Item 81)
  81. The pharmaceutical composition of item 79, wherein the ocular disease can comprise retinal neovascularization, choroidal neovascularization, and ocular tumor neovascularization.
(Item 82)
  81. The pharmaceutical composition of item 79, wherein the eye disease can comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 83)
  80. The pharmaceutical composition of item 79, wherein the eye disease can comprise corneal neovascularization and macular degeneration.
(Item 84)
  80. The pharmaceutical composition of item 79, wherein the composition is administered non-systemically to the eye of the subject.
(Item 85)
  A pharmaceutical composition for topical administration to the eye of a subject suffering from an eye disease, wherein the composition is in suspension, emulsion, or solution in the following:
  (A) CA4P in an amount ranging from about 0.1 mg / ml to about 100 mg / ml;
  (B) about 5 mg / ml carboxymethylcellulose; and
  (C) about 9 mg / ml NaCl,
A pharmaceutical composition comprising:
(Item 86)
  The composition has a final pH in the range of about 6.6 to 8.6, about 291 to 492m osmolality / kg H ₂ 86. The pharmaceutical composition of item 85, having an osmotic pressure in the range of O and a viscosity in the range of about 50 to 66 mPa · s.
(Item 87)
  86. The pharmaceutical composition of item 85, wherein the ocular disease can comprise retinal neovascularization, choroidal neovascularization, and ocular tumor neovascularization.
(Item 88)
  86. The pharmaceutical composition of item 85, wherein the eye disease can comprise diabetic retinopathy, retinopathy of prematurity, and retinoblastoma.
(Item 89)
  86. The pharmaceutical composition of item 85, wherein the eye disease can comprise corneal neovascularization and macular degeneration.
(Item 90)
  86. The pharmaceutical composition of item 85, wherein the composition is administered non-systemically to the eye of the subject.
(Item 91)
  A pharmaceutical composition for treating or preventing an eye disease and an eye tumor, said composition being pharmaceutically acceptable for administration to a subject in need of said treatment or prevention A pharmaceutical composition comprising a therapeutically effective amount of combretastatin A4 prodrug in combination with a carrier, excipient, diluent or adjuvant.
(Item 92)
  The therapeutically effective amount of combretastatin A4 prodrug is administered via intravitreal injection, subconjunctival injection, periocular injection, subthonone injection, by eye drops, by eye gel, by eye ointment, by iontophoresis, and 92. The pharmaceutical composition of item 91, administered non-systemically to the eye of said subject by an ocular implant and / or an ocular insert.
(Item 93)
  The therapeutically effective amount of combretastatin A4 prodrug is administered non-systemically to the eye of the subject in an amount of combretastatin A4 prodrug ranging from about 0.1 mg / ml to about 100 mg / ml. 92. The pharmaceutical composition according to item 91.
(Item 94)
  92. The pharmaceutical composition of item 91, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered systemically to said subject.
(Item 95)
  The therapeutically effective amount of combretastatin A4 prodrug is about 0.1 mg / m ² ~ About 120mg / m ² 92. The pharmaceutical composition of item 91, wherein the composition is administered systemically to said subject in an amount of combretastatin A4 prodrug in the range of

The invention will be better understood with reference to the accompanying drawings.
FIG. 1 is an anatomical view from the front and side of a mammalian eye. FIG. 2A shows a normal macular. FIG. 2B shows dry macular degeneration. FIG. 2C shows wet macular degeneration. 3A and 3B are magnified photographs of the corneal region showing vascular growth inhibition 28 days after CA4P administration, compared to vehicle control eyes. Figures 4A and 4B show microscopic histology of changes to the cornea (inhibition of blood vessel growth) 28 days after systemic administration of CA4P compared to vehicle control eyes. FIG. 5A shows the effect of a single dose of CA4P on ocular tumor neovascularization in an animal model of retinoblastoma. FIG. 5B shows the extent of tumor regression in an animal model of retinoblastoma following repeated doses of CA4P.

(Detailed description of the invention)
The human eye has several characteristics that are structurally unique: the human eye is exposed to the environment, severely weakened, has high blood flow in the choroid, and the anterior chamber and vitreous humor are completely Avascular and isolated from the circulatory system. The unusual structure of the eye provides ample opportunity for the delivery of tubulin binding agents by one or more non-systemic administration methods for the treatment of ocular conditions, diseases, tumors and disorders. A simplified anatomical view of the eye is shown in FIG.

  As previously described, neovascularization of ocular tissue is a pathogenic condition that occurs in various cancer diseases and is associated with varying degrees of visual dysfunction. The pathological control of neovascularization is potentially useful for patients suffering from diseases such as wet macular degeneration, proliferative diabetic retinopathy or premature retinopathy.

  Tubulin-binding drugs inhibit tubulin assembly by binding to tubulin-binding cofactors or cofactor-tubulin complexes in cells during mitosis and prevent division, thus proliferating cells. To prevent. Tubulin binding agents comprise a broad class of compounds that inhibit tubulin polymerization, which is generally a tumor-selective vascular target useful for cancer chemotherapy and other non-cancer applications (eg, eye diseases). It functions as a chemical factor.

  As noted above, one of the disadvantages of systemic administration of drugs to treat eye diseases is that systemic administration generally does not provide an effective level of drug specifically for the eye. Because systemically administered drugs can be metabolized in the body even before reaching the eye, high levels of drug may need to be administered to achieve effective intraocular concentrations . Non-systemic or local administration of the drug directly to the eye of a patient suffering from an eye disease allows for an effective concentration of the drug to be administered and is very beneficial to that patient.

  Eye signs that can be treated by non-systemic administration of tubulin binding agents according to the present invention include non-malignant vascular proliferative diseases characterized by neovascularization of the cornea, retina or choroid, as well as malignant vascular proliferative diseases ( For example, ocular tumors and cancers). Corneal neovascularization occurs in: Chlamydia trachomatis, virus-mediated keratitis, microbial keratoconjunctivitis, corneal transplants and burns. This includes infection (trachoma, herpes, leishmaniasis, onchocerosis, transplantation, burns (heat, alkali), trauma, nutritional deficiencies and contact lens induced damage. Retinal and / or corneal neovascularization is Occurs in macular degeneration, diabetic retinopathy and premature retinopathy Anterior neovascularization occurs in glaucoma.

  Non-systemic methods of administration of tubulin binding agents contemplated by the present invention include: intravitreal (injection), subconjunctival administration, periocular administration, subthonone injection, iontophoretic delivery, eye drops, gel Or via topical administration in ointments, and ocular inserts or implants.

  Tubulin binding agents can be administered intravitreally via direct injection into the vitreous humor of the eye. Tubulin binding agents can also be administered below the conjunctiva by subconjunctival injection and around the eye via periocular injection.

  Tubulin binding agents can also be administered by injection into the sub-Thonon space (under the Thonong sac) using a smooth tip Connor Canula. Using appropriate techniques, a medical professional administering a dosage of a tubulin binding agent may avoid puncturing the eye and damaging the optic nerve. After delivery, the injection site is paralyzed and this space serves as a reservoir for the drug. Subthononic administration is less invasive than vitreous injection.

  In another embodiment of the invention, the tubulin binding agent is biocompatible, biodegradable, and / or to provide sustained release of the drug and maintenance of a therapeutically effective drug concentration over time. Alternatively, it can be formulated as an ocular implant or insert containing a tubulin binding agent that is bioerodible. Drug-containing bioerodible ocular implants for implantation or insertion into a mammalian eye are described, for example, in US Pat. Nos. 5,904,144 and 5,766,242 (These are incorporated herein by reference in their entirety). An ocular implant generally includes a capsule that is placed at a desired location in the eye. The capsule may contain one or more medicaments or cells that produce biologically active molecules for continuous controlled delivery to the eye. The amount of drug that can be used in this embodiment will vary depending on the effective dose of the drug and the rate of release from the insert or implant on or in the eye.

  Since the sclera is exposed, an iontophoretic probe can be applied on the surface of the eye. Iontophoresis uses an electric current to drive the flow of ionic compounds across the cell membrane. This technology is currently utilized for transdermal delivery of ionic drugs. The two main mechanisms by which iontophoresis drives drug transport are: (a) iontophoresis (where charged ions are repelled from the same charge electrode), and (b) Electroosmosis (solvent convection through a charged “pore” in response to selective passage of counterions when an electric field is applied).

  Tubulin binding agents can also be formulated for topical administration to the eye in a sterile eye drop form.

  According to the present invention, a preferred tubulin binding agent is combretastatin A4 (“CA4”), a potential vascular targeting factor. CA4 is substantially insoluble in water. This feature interferes with the formulation of pharmaceutical preparations of this compound. Thus, a more preferred prodrug form of combretastatin A4 (“CA4P”) is utilized to correct the generally poor solubility of CA4. However, the present invention is not limited to this aspect, and CA4 formulations may work as well or better than CA4P.

  Combretastatin comes from the Combretastatin family of tropical and subtropical shrubs and trees, which represents a virtually unexplored reservoir of new materials with potentially useful biological properties. Lewy disease (see Watt et al., “The Medicinal and Poisonous Plants of Southern and East Africa”, E & S. Livingstone, Ltd., London, 1962, 194) (Combetrum tum. Illustrated is the genus Combretastatin with 25 species (10% of the total) known in primitive medical practice in Africa and India for a variety of uses as treatments.

  Combretastatin has been found to be an antitumor substance. Many combretastatins have been isolated, structurally solved, and synthesized. US Pat. Nos. 5,409,953 and 5,59,786 are referred to as A-1, A-2, A-3, B-1, B-2, B-3 and B-4. The isolation and synthesis of combretastatin is described. The disclosures of these patents are hereby incorporated by reference in their entirety. A related combretastatin (designated combretastatin A4) is described in US Pat. No. 4,996,237 to Pettit, which is incorporated herein by reference in its entirety.

  CA4P is a derivative of the natural combretastatin A4 subtype described in US Pat. No. 5,561,122, which is incorporated herein by reference. Preferred CA4P compounds substitute disodium phosphate for the -OH group in the CA4 structure, and this -OH group allows metabolic conversion of CA4P back to CA4 which is insoluble in water in vivo. However, the present invention is not limited to phosphate derivatives, and other prodrug moieties may replace the —OH group in the CA4 compound. It is further anticipated that phosphate prodrug salts other than the disodium salt of CA4P will be performed in substantially the same manner for the purposes of the present invention. Examples of other phosphate prodrug salts are described in PCT patent applications WO02 / 22626 and WO99 / 35150, the disclosures of which are incorporated herein.

  CA4P is the first in a new class of drugs (antitumor vascular targeting factors) that reduce solid tumors by selectively targeting and destroying tumor-specific blood vessels formed by angiogenesis . Anti-tumor vascular targeting and angiogenesis inhibition are related cancer therapies that fundamentally differ from conventional approaches to cancer treatment. In contrast to traditional methods, including direct attacks on cancer cells, these new drugs are the tumor life support system, a newly emerging network of blood vessels that form as a result of angiogenesis, previously Target budding of new blood vessels from existing blood vessels. Preclinical studies have shown that the use of these therapies can reduce and eventually eliminate tumors. Furthermore, when CA4P was used in animal cell models in vitro and in vivo, it showed significant specificity for vascular toxicity (Int. J. Radiat. Oncol. Biol. Phys. 42 (4): 895-903, 1998). Cancer Res. 57 (10): 1839-1834 1997).

  Both angiogenesis inhibitors and anti-tumor vascular targeting factors (eg combretastatin) target tumor blood vessels, but they differ in their approach and end result. For angiogenesis inhibition, the objective is to prevent tumor growth by inhibiting the formation of tumor-specific blood vessels that nourish and maintain the tumor. On the other hand, for anti-tumor vascular targeting, the goal is to remove and destroy tumors by selectively attacking and destroying existing blood vessels and creating rapid and irreversible deactivation of these blood vessels. Such an effect is not observed with anti-angiogenic agents. Only antivascular targeting activity can destroy existing blood vessels that support tumor growth. Combretastatin also has the ability to inhibit the proliferation of endothelial cells that produce and line new tumor vasculature (anti-angiogenic activity). Thus, it is believed that combretastatin can behave as both an anti-tumor vascular targeting factor and an anti-angiogenic agent. Preclinical studies have shown that both therapeutic agents leave blood vessels associated with unaffected normal tissue. The present invention contemplates the administration of CA4P administration alone and / or in combination with the current state of the pharmaceutical field for the treatment of eye diseases.

  The vasculature formed by angiogenesis has also been observed in diseases other than cancer, including ocular diseases such as macular degeneration, proliferative diabetic retinopathy and premature retinopathy. Preliminary studies to reduce such vasculature in an experimental eye model are described by Donald Armstrong, Ph. D. , D.C. Sc. , University of Florida, College of Veterinary Medicine, Division of Ophthalmology, he showed that CA4P accelerates the regression rate of preformed blood vessels in the eyes of experimental animal models. Figures 3A, 3B, 4A and 4B show the regression of preformed blood vessels in the rabbit eye studied in this experiment.

  CA4 and CA4P are ongoing clinical trials for the treatment of various diseases and symptoms, including use as anti-tumor vascular targeting factors and as inhibitors of angiogenesis. In addition, CA4P has shown the ability to treat ocular diseases such as subretinal neovascularization.

  The present invention also contemplates the use of synthetic analogues of combretastatin as described below: Bioorg. Med. Chem. Lett. 11 (2001) 871-874, 3073-3076, J. MoI. Med. Chem. (2002), 45: 1697-1711, WO 02/50007, WO 01/12579, WO 00/35865, WO 00/48590, WO 01/12579, US Pat. Nos. 5,525,632, 5,674,906 and the like. No. 5,731,353.

  Other tubulin binding agents that can be administered as VTAs include the following agents and their prodrugs: 2,3-disubstituted benzo [b] thiophenes (US Pat. No. 5,886,025; 6,162,930, and 6,350,777), 2,3-disubstituted benzo [b] furans (WO98 / 39332), 2,3-disubstituted indoles (WO01 / 19794), disubstituted Dihydronaphthalene (WO01 / 68654) or colchicine analog (WO99 / 02166). In addition, further non-cytotoxic prodrugs of vascular targeting factors, which are converted to substantially cytotoxic drugs by the action of endothelial enzymes selectively induced at enhanced levels at the site of vascular proliferation Is disclosed in WO 00/48606.

  Additional known tubulin binding agents that can be administered according to the present invention include: taxanes, vinblastine (vinca alkaloids), colchicine (colchicinoids), dolastatin, podophyllotoxin, steganacin, amphetinyl. , Flavonoids, lysoxine, curacin A, epothilone A, epothilone B, welwistatin, phenstatin, 2-strquinquinazolin-4 (3H) -one, stilbene 2-Aryl-1,8-naphthyridin-4 (1H) -one, and 5,6-dihydroindole (indolo) (2,1-a) isoquinoline.

  Regarding the administration and delivery of tubulin binding agents to the eye of a subject in need thereof, it is important to consider that the human eye has several structurally unique properties: When exposed to the environment, the human eye is severely debilitated, the human eye has high blood flow in the choroid, and the anterior chamber and vitreous humor are completely avascular and separated from the circulatory system. The unusual structure of the eye provides ample opportunities for alternative drug delivery methods. In this regard, four non-systemic modes of administration are contemplated by the present invention: intravitreal administration (injection), subthononic injection, iontophoretic delivery, implant / insert and ophthalmic delivery.

  Studies of ocular inflammation and biodistribution, and inhibition of blood vessel growth in animal models of cornea, choroid or retinal neovascularization after CA4P administration are described in the Examples section below.

Thus, angiogenic retinopathy and ocular tumors are themselves viable targets for CA4P treatment and other tubulin binding agents for a variety of reasons. Ie:
Tubulin-binding drugs can attack abnormal new blood vessels associated with retinopathy. This is because these blood vessels do not share structural similarity with BRB. Tubulin-binding drugs can stop the progression of diseases that mimic diseases in which blood vessels share structural similarities with solid tumor structures. In addition, tubulin binding agents can cause neovascular regression as observed in various preclinical studies.
• Since there is no 100% effective treatment for subretinal neovascularization, tubulin binding agents can be effective drugs when used in combination with the current state of treatment in the art.
• Currently the most improved treatment for retinopathy involves surgical intervention that may be painful and may require a long recovery period. Non-systemic or systemic administration of a tubulin binding agent is a non-surgical form of treatment.
CA4P, when delivered systemically or non-systemically, as a vascular targeting factor in animal models of corneal, retinal or choroidal neovascularization and in animal models with ocular tumors Indicates support.

  As described, CA4P and other vascular targeting factors and tubulin binding agents are delivered systemically in corneal, retinal or choroidal neovascularization and other disease and tumor models. Shows support. Preferred modes of systemic administration include parenteral administration and oral administration. Parenteral administration is a route of administration of a drug by injection under or through one or more skin layers or fascia. The parenteral route of administration by convention includes any route other than the oral-gastrointestinal tract (intestinal tract). Parenteral administration includes intravenous, intramuscular and subcutaneous routes.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Pharmaceutical compositions for topical ophthalmic administration include eye drops, eye drops gels, sprays, ointments, perfusions and inserts. Topical delivery formulations of tubulin binding agents should remain stable for long enough to achieve the desired therapeutic effect. In addition, this factor must penetrate the surface structure of the eye and accumulate in significant amounts at the site of the disease. Furthermore, local delivery agents should not cause excessive amounts of local toxicity.

  Ophthalmic solutions in the form of eye drops generally consist of an aqueous medium. Buffers, organic carriers, inorganic carriers, emulsifiers, wetting agents and the like can be added to accommodate a wide range of drugs with varying degrees of polarity. Pharmaceutically acceptable buffers for topical ophthalmic formulations include, among others, phosphate buffer, borate buffer, acetate buffer, glucuronate buffer, and the like. Drug carriers can include water, mixtures of lower alkanols and water, vegetable oils, polyalkylene glycols, petroleum-based jelly, ethyl cellulose, ethyl oleate, carboxymethyl cellulose, polyvinyl pyrrolidone, and isopropyl myristate. Ophthalmic sprays generally produce the same results as eye drops and can be formulated in a similar manner. Some ophthalmic drugs have poor permeability across the ocular barrier and cannot be administered as eye drops or sprays. Thus, ointments can be used to extend contact time and increase drug absorption. Continuous and constant perfusion with ophthalmic drug solutions can be achieved by placing polyethylene tubing into the conjunctival sac. The flow rate of the perfusate can be adjusted via a minipump system to produce continuous eye irrigation. The insert is generally placed in the upper conjunctival foramen, or less frequently, except that it is placed in the lower conjunctival sac rather than in the open cornea Similar to a soft contact lens placed on top. Inserts are generally made from biologically soluble materials that dissolve in tears or disintegrate simultaneously with the release of the drug.

In one embodiment, the active compound is coated on an implant or insert that is implanted in the eye. One example of such an implant contemplated by the present invention is Oculex Pharmaceuticals, Inc. , Sunnyvale, CA. The Oculex implant is a biodegradable BDD ^™ drug delivery device composed of a biodegradable microsize polymer system into which microencapsulated drug therapeutics can be implanted inside the eye. This implant releases the desired drug directly into the area of the eye that needs to be dosed over a predetermined period of time ranging from days to months to years.

It is especially advantageous to formulate topical compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dosage for the subject to be treated; each unit together with the required pharmaceutical carrier as desired. A predetermined amount of active compound calculated to produce a therapeutic effect. The specification of the dosage unit form of the present invention is due to the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technology of making such active compounds for the treatment of individuals. Shown and depends directly on these. Further known information regarding methods for producing formulations according to the present invention can be found, for example, in “Remington's Pharmaceutical Sciences”, Mack Publishing Co. ^{, Easter, PA, 15 th Ed} . (1975) can be found in standard references in the art.

  In addition to the non-systemic routes of administration discussed above, examples of systemic routes of administration include parenteral (eg, intravenous), intradermal, subcutaneous, oral (eg, inhalation), transmucosal Administration, and rectal administration. Solutions or suspensions used for parenteral or subcutaneous administration may include the following components: sterile diluents (eg, water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol) Or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; such as acetate, citrate or phosphate And buffers for adjusting isotonicity, such as sodium chloride or dextrose, pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, parenteral preparations Ampules, disposable syringes or multi-dose bottles made from plastic It may be enclosed in Le.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it can easily exit a syringe. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. For example, a carrier comprising water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof can be a solvent or dispersion medium. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents (eg, sugars), polyhydric alcohols (eg, mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions incorporate the active compound (eg, a vascular targeting agent) in the required amount, if necessary, in a suitable solvent (one or more combinations of the ingredients listed above), followed by sterilization. It can be prepared by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder for the preparation of a sterile injectable solution, the method of preparation is vacuum drying and lyophilization resulting in a powder of the active ingredient and any further desired ingredients from the previously sterile filtered solution.

  Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Here the compound in the liquid carrier is applied orally, swished by gargle, expelled or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients (eg starch Or lactose), disintegrants (eg, alginic acid, primogel, or corn starch); lubricants (eg, magnesium stearate or Sterote); glidants (eg, silicon dioxide colloids); sweeteners (eg, sucrose) Or saccharin); or a flavoring agent (eg, peppermint, methyl salicylate, or orange flavor).

  For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressurized container or dispenser (ie, a nebulizer) containing a suitable propellant (eg, a gas such as carbon dioxide).

  Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, surfactants, bile acids, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as generally known in the art.

  The compounds can also be prepared in the form of suppositories or retention enemas for rectal delivery (eg, using conventional suppository bases such as cocoa butter and other glycerides).

  In addition to the tubulin binding agents described above, the present invention also includes pharmaceutically acceptable carriers, diluents or excipients (eg, water, glucose, lactose, hydroxypropylmethylcellulose as well as those commonly known in the art. As well as other pharmaceutically acceptable carriers, diluents or excipients) and the use of pharmaceutical compositions and formulations containing tubulin binding agents.

As used herein, the term “pharmacologically effective amount”, “pharmaceutically effective dosage”, or “therapeutically effective amount” has been investigated by a researcher or clinician. Means the amount of a drug or agent that elicits a biological or medical response in a living tissue, system, animal or human. The dosage of CA4P for administration to the eye of a subject (non-systemic administration) is in the range of about 0.01 mg / ml to 100 mg / ml. The concentration of CA4P achieved in the eye should be therapeutically appropriate and is in the range of about 1 nanomolar to 100 millimolar. More preferred CA4P concentrations in the eye are in the range of about 1 micromolar to 100 micromolar. When CA4P is administered systemically, an amount of combretastatin A4 prodrug within the range of about 0.1 mg / m < ² > to about 120 mg / m < ² > is advantageously administered parenterally.

  Systemic and non-systemic administration of tubulin binding agents according to the present invention is intended to be formulated for administration to mammals, particularly humans. However, the invention is not limited in this respect and the formulations can be prepared according to veterinary guidelines for administration to animals as well.

  The invention will be further clarified by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both materials and methods, can be practiced without departing from the purpose and spirit of the invention.

Example 1.3 Study of eye irritation of CA4P and determination of mean acceptable dose (MTD) when administered topically to the eye using three routes of administration
(I) Intravitreal administration CA4P, a test article, was evaluated for its potential to cause intraocular irritation after intravitreal administration in rabbits. After general anesthesia, a 0.2 ml dose of CA4P was administered to the right eye of 8 rabbits. A 0.2 ml dose of 0.9% sodium chloride (USP) solution was administered to the left eye of these rabbits to serve as a negative control. Four different concentrations of CA4P were tested. Each of the four concentrations (0.1 mg / ml, 1.0 mg / ml, 10 mg / ml and 100 mg / ml) was dosed to the right eye of two rabbits. Approximately 48 hours after treatment, the eyes were examined using a biomicroscopic slit-lamp and an inverted ophthalmoscope. The score was recorded and the rabbit was euthanized. Immediately after euthanasia, a sample of the vitreous humor was removed and the white blood cell count determined using a hemocytometer. Numbers below 200 cells / mm ³ were considered acceptable.

The control eye did not change significantly in the eye tissue based on biomicroscopy and inversion ophthalmoscopic testing. For test treated eyes, evidence of irritation was observed in one animal at the 1 mg / ml concentration and for both at the 100 mg / ml concentration. The average number of cells in the vitreous humor is 9 cells / mm ³ for eyes given a concentration of 0.1 mg / ml; 962 cells / mm ³ for eyes given a concentration of 1 mg / ml; 10 mg 10 cells / mm ³ for eyes given a / ml concentration; 409 cells / mm ³ for eyes dosed with 100 mg / ml concentration and 5 cells / mm ³ for control eyes .

  Under study conditions, the controls responded as expected, with no significant responses in ocular testing and vitreous analysis. For the test treated animals, both animals dosed at a concentration of 100 mg / ml showed irritation and inflammation in both eye studies and evidence of inflammation from vitreous leukocyte analysis. For all of the low dose levels (0.1 mg / ml, 1.0 mg / ml and 10 mg / ml), there was no obvious evidence of irritation or inflammation.

(Ii) Administration under Thonon The test article CA4P was evaluated for primary eye irritation. Two 0.1 ml injections of appropriate CA4P concentrations (0.1 mg / ml, 1.0 mg / ml, 10 mg / ml and 100 mg / ml) were injected under the Tonon cavity of the right eye of two rabbits. A 0.2 ml portion of the buffered physiological saline solution was injected under the Thonon cavity of the left eye to serve as a negative control. Eye response was evaluated 24, 48 and 72 hours after sample instillation. On the third day, the rabbits were euthanized and their eyes were removed. Specimens were fixed, embedded, and histology was performed. Histopathological changes in ocular tissues were recorded. Emphasis was placed on testing for subthonon change.

  Under the conditions of this study, no irritation was observed in eyes treated with 0.1 mg / ml and 1.0 mg / ml concentrations compared to the corresponding negative control eyes. Slight irritation was observed in eyes treated with 10 mg / ml and 100 mg / ml concentrations compared to the corresponding negative control eyes.

(Iii) Topical drop
The test article CA4P was evaluated for primary eye irritation. A single 0.2 ml dose of CA4P dilution (0.1 mg / ml, 1.0 mg / ml, 10 mg / ml and 100 mg / ml) was placed in the lower conjunctival sac of the left eye and served as a comparative control. The contralateral eye was given a buffered saline solution. Eye response was evaluated 24, 48, and 72 hours after sample instillation.

  Under the conditions of this study, all visible responses of the test article dilution were considered insignificant compared to the control response. Microscopically, the test article was not considered a stimulant compared to the buffered saline solution control article.

  A summary of the results is shown in the following table:

（実施例２．異なる投与経路を用いて、眼に局所的に投与した場合の、ＣＡ４Ｐの体内分布の評価）
眼の新生血管形成の処置において有効であるために、非全身的な薬物投与方法は、眼の関連構造に浸透し、そして治療的に意味のある量の薬物を疾患部位に送達しなければならない。種々の非全身的注射の方法が、ＣＡ４Ｐの有意な体内分布を生じることを確認するために、放射標識された薬物の体内分布試験を実行した。

(Example 2. Evaluation of biodistribution of CA4P when administered locally to the eye using different administration routes)
To be effective in the treatment of ocular neovascularization, non-systemic drug administration methods must penetrate the relevant structures of the eye and deliver a therapeutically meaningful amount of the drug to the disease site . To confirm that the various non-systemic injection methods resulted in significant biodistribution of CA4P, biodistribution studies of radiolabeled drugs were performed.

(Method:)
Except as described below, the following experimental protocol was followed for each biodistribution.

¹⁴ C-CA4P (OXiGENE Inc., Watertown, Mass.) Was resuspended in 100 ul volume of saline and anesthetized male New Zealand rabbit (4 months old, 1.8-2.5 kg, n = 3 per sample) ) Was injected into the right eye using a 30G needle. Three different concentrations of ¹⁴ C-CA4P were tested (1, 10, 100 mg / ml) corresponding to the doses of 1, 5, and 5 uCi doses applied, respectively. A blank control group was also included. Rabbits were anesthetized at 1, 6, 24, and 48 hours and blood was collected. After blood collection, the animals were euthanized by Phenobarbital injection and the treated right eye was removed from all test animals. Eye tissue samples were dissected from the cornea, aqueous humor, vitreous, choroid, or retina, placed in 20 ml glass scintillation vials, vortexed, and incubated with 500 ul digestive fluid for 24 hours. Plasma was separated from whole blood by centrifugation (1,800 g for 10 minutes). Both ocular tissue samples and plasma were incubated at room temperature with 16 ml of Honic Fluor ^™ scintillation fluid for 24 hours before counting radioactivity. Each sample was counted for 5 minutes in a Betamatic V counter (Bio-Tek Kontron Instruments, St Quentin en Yvelines, France). Counts per minute ( "cpm"), quench curves for the conversion to collapse per minute ( "dpm"), from each of the blank matrix spiked with calibration curves and ^{14 C} standard was obtained from ^{14 C} Standard Was automatically executed by the β counter. Drug concentration was calculated from CA4P (tissue ng-Eq / g) ng equivalent (measured dpm value, tissue specimen weight, and drug specific activity (0.37 mCi / mg), then control ocular tissue The corresponding background was subtracted). The tissue concentration of 1 uM CA4P is equal to 440 nEq / tissue.

(result:)
(I) Intravitreal administration Table 1 shows the results of biodistribution after intravitreal administration. In all experimental tissues, the degree of ocular penetration was dependent on the CA4P concentration placed on the surface of the eye. The highest drug concentration in the eye (“C _max ”) was achieved within 1 hour after administration. Drug-related concentrations of drug (> 1 uM) were delivered to the retina at all tested concentrations. High concentrations of drug were also found in the vitreous and sclera. Relatively little drug was found in ocular aqueous humor or plasma.

  (Table 1: Biodistribution of CA4P after intravitreal injection)

（ｉｉ）トノン下（ｓｕｂ−Ｔｅｎｏｎ）投与
（１）体内分布
表２は、トノン下注射後の体内分布の結果を示す。全ての実験した組織において、眼浸透の程度は、眼の表面に配置されたＣＡ４Ｐ濃度に依存した。眼において最も高い薬物濃度は、投与後１時間以内であった。薬物の治療関連濃度（＞１ｕＭ）は、１００ｍｇ／ｍｌおよび１０ｍｇ／ｍｌの投与用量で、網膜および脈絡膜に送達された。高濃度の薬物もまた、強膜において見出された。硝子体、房水または血漿において、薬物は相対的にほとんど見出されなかった。

(Ii) Sub-Thonon administration (1) Biodistribution Table 2 shows the results of biodistribution after subtonon injection. In all experimental tissues, the degree of ocular penetration was dependent on the CA4P concentration placed on the surface of the eye. The highest drug concentration in the eye was within 1 hour after administration. Drug-related concentrations of drug (> 1 uM) were delivered to the retina and choroid at doses of 100 mg / ml and 10 mg / ml. High concentrations of drug were also found in the sclera. Relatively little drug was found in the vitreous, aqueous humor or plasma.

  (Table 2: Biodistribution of CA4P after injection under tononone)

（ｉｉｉ）結膜下投与
結膜下注射を、０．１、１、１０および１００ｍｇ／ｍｌの用量で投与した。^１４Ｃ−ＣＡ４Ｐ溶液を、０．２４％ １０Ｎ ＫＯＨおよび０．０１％塩化ベンザルコニウムと共に処方し、そして１００ｕｌの容量で注射した。動物を、右眼の単回結膜下注射により、５ｕＣｉの適用用量で処置した。送達後、眼を２〜５秒間、穏やかに閉じさせた。以下の表３に、実験の結果を示す。

(Iii) Subconjunctival administration Subconjunctival injections were administered at doses of 0.1, 1, 10 and 100 mg / ml. ¹⁴ C-CA4P solution was formulated with 0.24% 10N KOH and 0.01% benzalkonium chloride and injected in a volume of 100 ul. The animals were treated with an applied dose of 5 uCi by a single subconjunctival injection in the right eye. After delivery, the eyes were gently closed for 2-5 seconds. Table 3 below shows the results of the experiment.

  The highest drug concentration in the eye was within 1 hour after administration. Drug-related concentrations of drug (> 1 uM) were delivered to the cornea, retina and choroid at doses of 100, 10 and 1 mg / ml. In the vitreous or plasma, relatively few drugs were found.

  (Table 3: CA4P biodistribution after subconjunctival injection)

（ｉｖ）眼周囲投与
表４は、眼周囲注射後の体内分布の結果を示す。試験した全ての組織において、眼浸透の程度は、その眼の表面上に配置されたＣＡ４Ｐの濃度に依存した。眼内の薬物の最高濃度は、投与後の最初の１時間内にあった。治療学的に適切な濃度（＞１μＭ）の薬物を、全ての投与用量にて、網膜および脈絡膜に送達した。高濃度の薬物はまた、強膜においても観察された。相対的に少量の薬物が、硝子体または血漿において見出された。

(Iv) Periocular administration Table 4 shows the results of biodistribution after periocular injection. In all tissues tested, the degree of ocular penetration was dependent on the concentration of CA4P placed on the surface of the eye. The highest concentration of drug in the eye was within the first hour after administration. A therapeutically relevant concentration (> 1 μM) of drug was delivered to the retina and choroid at all doses administered. High concentrations of drug were also observed in the sclera. A relatively small amount of drug was found in the vitreous or plasma.

  (Table 4: Distribution of CA4P in the body after periocular injection)

（ｖ）局所処方物
局所ゲルおよび局所溶液を、眼の表面へのＣＡ４Ｐの局所送達に適切な局所処方物として使用するために開発した。局所溶液（１％、３％、および１０％）を、０．９％ ＮａＣｌ（Ａｇｕｅｔｔａｎｔ，Ｌｙｏｎ，Ｆｒａｎｃｅ）中に直接調製し、そして０．２μｍのフィルターを用いて滅菌した（ｐＨ：６．４〜８．５、浸透圧モル濃度：２９０〜４５９ｍｏｓｍｏｌ／ｋｇ Ｈ_２Ｏ）。低粘度の局所ゲル（１％、３％、および１０％）を、０．９％ ＮａＣｌを用いて、０．５％カルボキシメチルセルロース（Ｓｉｇｍａ Ａｌｄｒｉｃｈ Ｃｈｉｍｉｅ，Ｓｔ．Ｑｕｅｎｔｉｎ Ｆａｌｌａｖｉｅｒ Ｃｅｄｅｘ，Ｆｒａｎｃｅ）中で調製した。各ゲルの物理化学的仕様を、表５に示す。

(V) Topical formulations Topical gels and solutions were developed for use as topical formulations suitable for local delivery of CA4P to the ocular surface. Topical solutions (1%, 3%, and 10%) were prepared directly in 0.9% NaCl (Aguettant, Lyon, France) and sterilized using a 0.2 μm filter (pH: 6.4). 8.5, _{osmolarity: 290~459mosmol / kg H 2 O)} . Low viscosity topical gels (1%, 3%, and 10%) were prepared in 0.5% carboxymethylcellulose (Sigma Aldrich Chimie, St. Quentin Fallavier Cedex, France) with 0.9% NaCl. . Table 5 shows the physicochemical specifications of each gel.

  (Table 5: Topical CA4P gel formulation)

局所処方物を、５０μｌの容量中、５μＣｉの適用用量にて、右眼表面に適用した。強膜の代わりに、角膜をサンプリングした。サンプルを、０．５時間、１．６時間、および２４時間で採取した。

The topical formulation was applied to the right eye surface at an applied dose of 5 μCi in a volume of 50 μl. The cornea was sampled instead of the sclera. Samples were taken at 0.5 hours, 1.6 hours, and 24 hours.

  Table 6 shows the biodistribution results after administration of each topical CA4P gel formulation. In all tissues tested, the degree of ocular penetration was dependent on the concentration of CA4P in each gel formulation. The highest concentration of drug in the eye was within the first hour after administration. A therapeutically relevant concentration (> 1 μM) of drug was delivered to the cornea, retina and choroid using all three gel formulations. A relatively small amount of drug was found in plasma.

  (Table 6: CA4P biodistribution after topical administration of gel)

表７は、各局所ＣＡ４Ｐ溶液処方物の投与後の体内分布の結果を示す。試験した全ての組織において、眼浸透の程度は、各溶液処方物中のＣＡ４Ｐの濃度に依存した。眼内の薬物の最高濃度は、投与後の最初の１時間内にあった。治療学的に適切な濃度（＞１μＭ）の薬物を、３つ全ての溶液処方物を用いて角膜に送達し、一方、１０ｍｇ／ｍｌ用量が、網膜および脈絡膜への、有意な量の薬物送達を生じた。

Table 7 shows the biodistribution results after administration of each topical CA4P solution formulation. In all tissues tested, the degree of ocular penetration was dependent on the concentration of CA4P in each solution formulation. The highest concentration of drug in the eye was within the first hour after administration. A therapeutically relevant concentration (> 1 μM) of drug is delivered to the cornea using all three solution formulations, while a 10 mg / ml dose delivers a significant amount of drug to the retina and choroid Produced.

  (Table 7. CA4P biodistribution after topical administration of solution formulation)

これらの試験から、任意の種々の方法によるＣＡ４Ｐの非全身送達は、角膜、網膜、または脈絡膜において治療学的に適切な薬物濃度を達成するのに有効であることが明らかである。これらの組織の各々は、眼の潜在的な新生血管形成部位である。

From these studies, it is clear that non-systemic delivery of CA4P by any of a variety of methods is effective in achieving therapeutically relevant drug concentrations in the cornea, retina, or choroid. Each of these tissues is a potential neovascularization site in the eye.

(Example 3. Ocular administration of CA4P by ion osmosis)
CA4P is ionizable at physiological pH and therefore follows ion osmotic delivery. The effectiveness of transcranial ion osmotic delivery of CA4P was demonstrated with a 180 μl silicone receptacle shell lined with a silver chloride coated silver foil current distribution component, a connector lead wire, and a monolayer of hydrogel impregnated polyvinyl acetal matrix ( Ocular rabbit ophtalmic applicator (IOMED) composed of CA4P (10 mg / ml))
Inc. , Salt Lake City, UT). The contact surface area of the applicator was 0.54 cm ² . The applicator was placed on the edge (superior cul-de-sac) of the New Zealand white rabbit (3-3-3. 5 kg, for each treatment, n = 6) was placed in the sclera of the right eye. DC anodic ion infiltration was performed at 2 mA, 3 mA, and 4 mA for 20 minutes using a Phoresor II ^™ PM700 (IOMED Inc., Salt
(Lake City, UT) was performed with each applicator using a power supply. Passive ion infiltration (20 mA for 20 minutes) was used as a control. After treatment, the animals were euthanized and the eyes were removed 30 minutes after treatment, rinsed with tap water, and frozen at -70 ° C. Retinal and choroidal tissues were examined from these samples.

  CP4A, CA, and internal standard diethylstilbestrol (Sigma Chemical Company) were quantified from approximately 100 mg of tissue using chromatographic tandem mass spectrometry (“LC / MS / MS”). An aliquot of the methanol extract was injected into a SCIEX APIO 3000 LC / MS / MS instrument equipped with an HPLC column. The peak areas of m / z 315 → 285 product ion of CA43 and m / z 395 → 79 product ion of CA4P were measured against the peak area of m / z 267 → 237 product ion of the internal standard. Quantification was performed using a weighted (1 / X) linear least squares regression analysis generated from an enhanced calibration standard prepared just before each run. This initial combretastatin amount was significantly greater than the quantification range and therefore had to be extrapolated after analysis. As Table 8 shows, the delivery of total combretastatin to the choroid and retina is increased approximately 15-fold by iontophoresis when compared to passive delivery. These levels represent thousands of times over the concentration (2-3 μM) considered to be a therapeutically relevant concentration to inhibit tubulin binding. However, this retina / choroid delivery did not appear to be current dependent.

  (Table 8. Enhanced ion permeation of combretastatin delivery to the retina / choroid (mean ± standard deviation))

（実施例４．ＣＡ４Ｐの全身投与による角膜の新生血管形成の処置）
病原性の眼の新脈管形成を刺激するために、眼の新生血管形成を、３０μｇの投薬量にて、ウサギの眼に角膜内注射によって脂質ヒドロペルオキシド（ＬＨＰ）を投与することによって、誘導した。数日〜１４日後に、眼血管が、ＬＨＰ侵襲（ｉｎｓｕｌｔ）に起因して、この注射された眼に形成された。被験体を、２つの群に分けた；１つの群の被験体は、５日間にわたる１日１回、４０ｍｇ／ｋｇの投薬量での静脈内投与による、所定のコンブレタスタチンＡ４リン酸二ナトリウムを受け、一方、コンブレタスタチンＡ４リン酸二ナトリウムを含まないビヒクルを、同じ期間にわたる水の投薬としての静脈内投与によって、他の群に投与した。両方の群の眼を、７日後に試験した。コンブレタスタチンＡ４リン酸二ナトリウムで処置した群において、４０％以上の血管の減少が観察されたが、他の群においては、観察されなかった。

Example 4. Treatment of corneal neovascularization by systemic administration of CA4P
In order to stimulate angiogenesis of pathogenic eyes, ocular neovascularization is induced by administering lipid hydroperoxide (LHP) by intracorneal injection to rabbit eyes at a dosage of 30 μg. did. Several days to 14 days later, ocular blood vessels were formed in the injected eye due to LHP insult. Subjects were divided into two groups; one group of subjects was given combretastatin A4 disodium phosphate by intravenous administration at a dosage of 40 mg / kg once a day for 5 days. On the other hand, vehicle without combretastatin A4 disodium phosphate was administered to other groups by intravenous administration as a water dosing over the same period. Both groups of eyes were examined after 7 days. In the group treated with combretastatin A4 disodium phosphate, a vascular reduction of more than 40% was observed, but not in the other groups.

Example 5. Treatment of corneal neovascularization by systemic administration of CA4P
To assess the ability of CA4P to inhibit corneal neovascularization, a rabbit cornea model was used. In this model, neovascularization was induced by linoleic acid hydroperoxide (“LHP”) injection (Ueda et al., Angiogenesis, 1997, 1: 174-184). Injection of LHP in the corneal stroma stimulates the localized production of angiogenic cytokines within this cornea. Circumlimal plexus vessels responded to angiogenic stimuli by moving towards the LHP injection site. The therapeutic effect of systemically delivered CA4P was assessed by measuring the length of these growing blood vessels.

(experimental method)
As outlined in Table 9 below, adult male New York rabbits (2.7-3.0 kg) were injected 5 μm from the upper edge with a 10 μl suspension of LHP (60 μg) to obtain corneal Angiogenesis was induced. The blood vessels grew at a rate of 0.25 mm / day. Groups 2 and 4 were injected intraperitoneally (“IP”) with CA4P (50 mg / kg) 3 and 10 days after blood vessel growth, respectively. Treatment groups 1 and 3 were injected with saline control on days 3 and 10 after LHP injection. The cornea surface photographs were taken on the 0th, 3rd, 6th, 12th, 17th, and 28th days after LHP injection. After each photograph, corneal vessels were observed under a surgical microscope and the most prominent vessel length was measured using a Castroviejo caliper.

  In addition to measuring the longest vessels, histological analysis was performed on day 28 to assess the amount of lysed extracellular matrix, vessel wall thickness, and the degree of vessel branching. The eyeball is removed from the euthanized animal and its vitreous is removed from each eye and then fixed with 4% paraformaldehyde for 45 minutes and with 0.2 M cacodylate buffer (pH 7.4) overnight. did. Eyes were embedded in paraffin, cut to 3 μm thickness and stained with hemolysin and eosin.

  (Table 9. Experimental design)

（結果）
表１０および表１１は、処置の介入時間および処置回数の関数として、血管の長さに対するＣＡ４Ｐの効果を要約している。ＣＡ４Ｐ処置を用いて、最初の脈管刺激から３日以内に介入した場合（表１０、群２）、薬物は、新生脈管成長の完全な阻害を引き起こした。対照的に、ビヒクルコントロール群における血管は、成長し続けた。この効果を、脈管形成阻害または脈管形成効果として定性化し得る。ＣＡ４Ｐ処置を用いて、脈管刺激の１０日後に介入した場合（表１１、群２）、その効果は、同じであった。

(result)
Tables 10 and 11 summarize the effect of CA4P on vessel length as a function of treatment intervention time and number of treatments. When CA4P treatment was used to intervene within 3 days of initial vascular stimulation (Table 10, Group 2), the drug caused complete inhibition of neovascular growth. In contrast, blood vessels in the vehicle control group continued to grow. This effect can be qualified as angiogenesis inhibition or angiogenesis effects. When CA4P treatment was used to intervene 10 days after vascular stimulation (Table 11, group 2), the effect was the same.

  (Table 10: Initial intervention: treatment begins on day 3)

（表１１：後期介入：１０日目に処置を開始する）

(Table 11: Late intervention: treatment begins on day 10)

図３Ｂは、２８日目でのＣＡ４Ｐで処置した眼の表面写真を示す。この写真は、図３Ａに示したビヒクルコントロールの眼と比較した、ＣＡ４Ｐ投与後２８日目における血管成長の阻害をさらに示す。

FIG. 3B shows a surface photograph of an eye treated with CA4P on day 28. The photograph further shows inhibition of blood vessel growth at 28 days after CA4P administration, compared to the vehicle control eye shown in FIG. 3A.

  The micrographs (400 × magnification) shown in FIGS. 4A and 4B are examples of stained histochemical specimens obtained from the same sample on day 28. In animals treated with vehicle (FIG. 4A), blood vessels appeared round and numerous. In contrast, in animals treated with CA4P (FIG. 4B), blood vessels appeared narrower and fewer. In addition, evidence of vascular regression was observed during the late phase of CA4P intervention (data not shown). CA4P could reduce the width of established blood vessels and seemed to be able to significantly inhibit branch sprouting from the blood vessels. This indicates an additional vascular targeting effect.

Example 6. Treatment of choroidal neovascularization in an animal model of macular degeneration by systemic administration of CA4P
Choroidal neovascularization is a major cause of severe visual loss in patients with age-related macular degeneration or wet macular degeneration. To investigate the ability of CA4P to inhibit blood vessel growth in the choroid, a mouse model of choroidal neovascularization was tested. In this model, researchers created a wound on the Bruch membrane of C57BL / 6J mice using a krypton laser. Each eye suffered some burns. This burn induced a classic wound healing response that induced neovascularization within the choroid. This krypton laser photocoagulation method is described by Tobe et al., Am. J. et al. of Pathology, 1998, 153 (5): 1641-6. In a subset of animals (n = 19), CA4P was administered systemically by IP injection at a dose of 100 mg / kg / day. Histopathology and fluorescein angiography were used to identify neovascularization around the burn. Electron microscopy was used to measure the lumen diameter of fenestrated neovascularization within choroidal neovascular injury. Table 12 shows the results of CA4P treatment versus mean vascular lumen area. Animals treated with CA4P had approximately 50% less vascular lumen area (mm ² ) when compared to saline treated animals (n = 33). Statistical analysis of these results showed a high degree of significance.

  Table 12. Mean luminal area of mice treated with CA4P and mice treated with vehicle

（実施例７．ＣＡ４Ｐの全身投与による、未熟児網膜症のマウスモデルにおける網膜の新生血管形成の処置）
哺乳動物の眼の内部網膜は、表在網膜の毛細血管床から酸素を受け取る。この毛細血管床は、内境界膜の下に位置され、この膜は、内部網膜と外部の無血管硝子体との間で境界面として働く。網膜の新生血管形成および網膜症の病理は、虚血（これは、網膜の内境界膜を越えて硝子体内に、新生血管形成の成長を誘導する）から生じ、重篤な視覚損失を引き起こし、しばしば網膜剥離の原因となる。酸素誘導性網膜新生血管形成の十分に特徴付けされたマウスモデルは、早熟で産まれた乳児によって示される未熟児網膜症（「ＲＯＰ」）を綿密に模倣し、種々の他の虚血誘導性未児網膜症（糖尿病性網膜症が挙げられる）に共通な特徴を示す（Ｓｍｉｔｈら、Ｉｎｖｅｓｔ．Ｏｐｈｔｈａｌｍｏｌ．Ｖｉｓ．Ｓｃｉ，１９９４，３５：１０１−１１）。このモデルにおいて、新生期のマウスを、持続性高酸素条件下（７５％酸素で７日間）に曝した。この条件は、表在網膜の毛細血管床の発達を阻害する。マウスの子を、純粋な酸素環境から離し、環境酸素の相対的低酸素中に置いた場合、発達不十分な表在網膜の毛細血管床は、十分な量の酸素を角膜に送達することができない。この網膜は、重篤な病理学的結果を引き起こす脈管形成サイトカインを生成することによって、酸素不足に応答する。脈管形成サイトカインの局在生成は、発達不十分の表在網膜の毛細血管床が、内境界膜を破壊する新たな血管を出芽するのを引き起こし得る。硝子体における異常な血管の成長は、重篤な瘢痕組織の形成および牽引誘導性網膜剥離を引き起こす。

Example 7. Treatment of retinal neovascularization in a mouse model of retinopathy of prematurity by systemic administration of CA4P
The internal retina of a mammalian eye receives oxygen from the capillary bed of the superficial retina. This capillary bed is located below the inner limiting membrane, which acts as a boundary between the inner retina and the outer avascular vitreous. Retinal neovascularization and retinopathy pathology results from ischemia (which induces neovascularization growth into the vitreous beyond the inner lining of the retina), causing severe visual loss, Often causes retinal detachment. A well-characterized mouse model of oxygen-induced retinal neovascularization closely mimics retinopathy of prematurity (“ROP”) exhibited by prematurely born infants, and various other ischemia-induced It shows features common to retinopathy of infants, including diabetic retinopathy (Smith et al., Invest. Ophthalmol. Vis. Sci, 1994, 35: 101-11). In this model, neonatal mice were exposed to sustained hyperoxic conditions (75% oxygen for 7 days). This condition inhibits the development of the superficial retinal capillary bed. When a mouse pup is placed away from a pure oxygen environment and placed in a relative hypoxic environment, the underdeveloped superficial retinal capillary bed can deliver a sufficient amount of oxygen to the cornea. Can not. The retina responds to hypoxia by producing angiogenic cytokines that cause severe pathological consequences. Localization of angiogenic cytokines can cause underdeveloped superficial retinal capillary beds to sprouting new blood vessels that destroy the inner limiting membrane. Abnormal blood vessel growth in the vitreous causes severe scar tissue formation and traction-induced retinal detachment.

  Treating neonatal mice with CA4P immediately after removal from hyperoxic conditions is expected to be an effective treatment method for ROP. Retinal neovascularization is performed in accordance with existing methods (Majka et al., Invest. Ophthalmol. Vis, Sci. 2001, 42: 210-15) of penetrating endothelial cells in retinal tissue sections of treated and untreated eyes. It can be quantified by counting the number of chemically stained nuclei. The number of nuclei penetrating the inner limiting membrane is expected to be significantly reduced in CA4P eyes.

Example 8. Treatment of ocular tumors in a mouse model of retinoblastoma by subconjunctival administration of CA4P
A mouse transgenic model of retinoblastoma was used. In this model, SV-40 large T antigen positive mice develop left and right retinoblastoma resembling human pediatric retinoblastoma. In this model, tumors initially appear at 4 weeks of age and develop in a stable and reproducible manner (Hayden et al., Arch Ophthalmol. 2002; 120 (3): 353-9). In one experiment, 12 week old animals (n = 48) were treated with a single 20 μl subconjunctival injection (100 mg / ml) of CA4P in the right eye only. Control mice (n = 8) were treated with balanced salt solution (“BSS”). Eyes were sampled by removal on days 1, 3, 7, 14, 21, and 28 after treatment (n = 8 (eye / sampling)). Samples were fixed, embedded in paraffin, serially cut and prestained with hematoxylin, eosin, and PAS. Each tissue sample was tested for histopathological tumor vascular response. As illustrated in FIG. 5A, a significant decrease in intratumoral neovascularization was seen on days 1, 3, and 7.

  In separate dosing experiments, animals (n = 48) were treated every other week at a concentration of 100 mg / ml, 10 mg / ml, 1 mg / ml, 0.1 mg / ml or 0.01 mg / ml in a volume of 20 μl. Treated with two consecutive subconjunctival injections of CA4P (n = 6 (eye / sampling)). The control group (n = 6) received continuous subconjunctival injections of BSS. Eyes were removed 28 days after treatment and tested for reduction in tumor volume. FIG. 5B illustrates the dose-dependent effect of CA4P on tumor vascular volume compared to control. Intratumoral vascularity did not exist at treatment dose levels higher than 10 mg / ml, and no evidence of toxicity was noted at any treatment dose at any time.

(Other embodiments)
While the invention has been described in conjunction with the detailed description thereof, it is to be understood that the foregoing description is intended to be illustrative and not intended to limit the scope of the invention, The invention is defined by the scope of the appended claims.

  It should also be understood that the drawings are not necessarily drawn to scale, but are merely conceptual in nature.

Claims (1)

The invention described herein.

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