RU2354398C2 - Compositions and methods for insertion of tubulin-binding agents for eye disease therapy - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present group of inventions refers to medicine, namely to ophthalmology, and involves therapy of eye diseases caused by nonmalignant neovascularisation, and also a medical product and pharmaceutical compositions containing prodrug combretastatin A4.

EFFECT: inventions provide effective therapy ensured with ability of prodrug combretastatin A4 to bind tubulin thereby preventing pathological vessel proliferation.

29 cl, 12 tbl, 5 dwg, 8 ex

Description

Field of Invention

The invention relates to the administration of vascular-targeted agents, in particular tubulin-binding agents, for treating eye diseases.

BACKGROUND OF THE INVENTION

The eye is fundamentally one of the most important organs throughout life. Due to aging, diseases and other factors that can adversely affect vision, the ability to maintain healthy eyes becomes very important. The main cause of blindness is the inability to administer drugs or therapeutic agents to the eye and to maintain said drugs or agents in a therapeutically effective concentration in it. Taking the drug inside or injecting the drug in a place other than the eye ensures systemic delivery of the drug. However, such systemic administration does not provide effective levels of the drug in the eye and, therefore, may necessitate the administration of often unacceptably high levels of the drug to achieve effective intraocular concentrations.

The macula is a region of the retina that contains an increased concentration of photosensitive cells that are responsible for clear detailed vision (a generalized anatomical graphic image of the human eye is shown in figure 1). Macular degeneration is an inaccurate historical name given to an under-understood group of diseases that cause loss of function of photosensitive macula cells. Macular degeneration results in the loss of vital central vision and detailed vision. Patients affected by macular degeneration perceive a blank spot in the center of their field of vision and often lose the ability to read small print (source: Macular Degeneration 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 will be affected by macular degeneration, and the incidence of the disease increases with age. An estimated 1.2 million out of 12 million people with macular degeneration will suffer severe central vision loss each year. Each year, 200,000 individuals lose their central vision completely in one or both eyes.

Although the exact cause of macular degeneration is unknown, the architecture of macula reveals the cause of how the disease can be initiated. The macula contains highly active photoreceptors that consume large amounts of energy. Generating this energy requires a rich supply of oxygen and nutrients. A macula lutea has one of the highest blood flow velocities through the vessels supplying it (also known as the choroid itself). Anything that impedes this rich blood supply can cause macular dysfunction. An oxygen-deprived macula reacts with the production of cytokines, which signal the growth of endothelial cells and the growth of new vessels (neovascularization).

There are two main types of macular degeneration: dry form and wet form. Approximately 85% to 90% of macular degeneration cases are of the dry type. In the dry form of the disease, retinal damage is associated with the formation of yellow deposits under the macula, known as drusen. Druze deposition correlates with a decrease in the thickness of the retinal cells that the macula contains. The degree of loss of central vision is directly related to the localization and severity of retinal thinning caused by the Druze. 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 macular degeneration. In a small percentage of people with a dry form of macular degeneration, the disease progresses to a wet form of macular degeneration. Figure 2 shows a healthy macula and a dry form of macular degeneration.

The wet form of macular degeneration is a rapidly progressive disease that almost always leads to severe vision loss. The loss of vision associated with wet macular degeneration is the result of subcellular neovascularization. The rapid growth of the retina blood vessels causes the retinal cells lying on top of the layer to bend, and it becomes detached from the nutrient-rich choroid of the eye. In extreme cases of wet macular degeneration, proliferating vessels penetrate the retina and infiltrate the vitreous. There are several treatments for wet macular degeneration, but none of them give long-term satisfactory results. Figure 3 shows a healthy macula and a wet form of macular degeneration.

Modern standard treatment for macular degeneration is laser photocoagulation. An ophthalmologist performing laser photocoagulation determines the localization of aberrant vessels by fluorescence angiography and selectively cauterizes the vessels by laser removal. A side effect of laser surgery is the destruction of the retinal layer lying directly on top of the aberrant vessels. Patients treated with laser photocoagulation have a pronounced loss of vision immediately after treatment, and this represents an unacceptable negative side effect. In general, laser surgery is seen as a palliative treatment that is only moderately effective in terms of slowing the development of the disease.

Photodynamic therapy is the current state of treatment for macular degeneration. The US Food and Drug Administration has approved Verteporfin for Injection (Visudyne ™, developed by Ciba Vision & QLT) for the treatment of wet age-related macular degeneration. A patient receiving photodynamic therapy is injected with a photoreactive compound (verteporfin) and immediately treated with a non-destructive eye laser. The ophthalmologist performing the operation identifies aberrant vessels and directs the laser beam in the direction of the aberrant vessels. Verteporfin, when activated by a laser, generates a transient burst of energy that effectively burns out any cells within close proximity to the activated molecule (Source: HHS News, U.S. Dept. Of Health and Human Services, April 13, 2000).

Ionizing radiation is used to destroy proliferating vessels (proliferating cells are more sensitive to radiation than dormant cells). Ionizing radiation is usually supplied by a beam that is large enough to come in contact with as much of the eye as possible. In 1993, a group at the University of Belfast in Northern Ireland reported that they had tested the use of X-rays in a small number of patients with a wet form of macular degeneration. Their positive results have been confirmed by several similar studies of the use of x-rays carried out by other research groups in Europe.

Another disabling eye disease is preterm retinopathy (ROP). ROP is an ocular disease that occurs in a significant percentage of premature babies. The last 12 weeks of full-term pregnancy (from 28 to 40 weeks) are especially active months in the development of the fetal eye. Prenatal development of blood supply to the retina (the choroid proper) begins in the optic nerve at week 16 and progresses with a radial type in the direction of the anterior region of the retina before birth (40 weeks). If the birth is premature, the retinal vasculature does not have enough time for full development, and the front edges of the retina become oxygen-free. The lack of oxygenation in the anterior retina is the underlying cause of ROP. (Source: The Association of Retinopathy and Related Diseases, Franklin, MI).

In premature infants, a significant portion of the anterior retina is deprived of adequate blood supply. Deprived of oxygen, the anterior part of the retina responds by signaling to the growth of new vessels. Pathological neovascularization in the area between the anterior and posterior retina initiates a cascade of events with severe pathological consequences. As new vessels grow in response to chemical signals, arteriovenous shunts form in the area between the posterior retina and the avascular anterior retina. These vascular shunts gradually increase, becoming thicker and more elevated. New vessels are accompanied by infiltrating fibroblasts, which form fibrous scar tissue. Ultimately, a ring of scar tissue forms, which is attached to the retina, as well as to the vitreous gel. A ring of scar tissue can extend 360 degrees around the inside of the eye. When the specified scar tissue contracts, it pulls the retina and creates a retinal detachment. If enough scar tissue forms, the retina may become completely detached. Premature infants are at risk of developing ROP because they have been removed from the protective environment of the uterus and are exposed to many angiogenic stimuli, including drugs, high oxygen levels, and changes in lighting and temperature. Some of all these factors can influence the development of ROP. Fortunately, most premature babies do not develop ROP, and most babies with ROP experience spontaneous improvement. If ROP develops, it usually occurs between the 34th and 40th weeks after conception, regardless of the gestational age at birth.

It has been shown that a technique called cryotherapy has a beneficial effect in the treatment of ROP. Cryotherapy involves placing a probe having a temperature below zero on the outer wall of the eye (sclera). The probe causes a crystallization zone to form on the surface of the retina between the sclera and the vitreous. For the treatment of the entire avascular region, which is located in front of the crest of the new vessels, many cryotherapy sessions are performed. Treatment of the ridge itself is avoided since the ridge has a tendency to bleed and can cause vitreous hemorrhage when frozen.

The mechanism of action of cryotherapy is not fully understood. The working hypothesis is that cryotherapy is likely to damage the avascular layer of the anterior retina. This damage leads to thinning of the retina, which provides the possibility of facilitated diffusion of oxygen into the remaining viable cells. In addition, in the retina, subjected to cryotherapy, there are fewer viable cells and, consequently, reduced oxygen demand. Reduced oxygen demand dulls angiogenic stimuli and stops neovascularization. Cryotherapy has been found to reduce the risk of retinal detachment from 43% in untreated eyes to 21% in treated eyes. However, cryotherapy has possible complications; The procedure is often performed under general anesthesia, which can be risky for premature babies.

Laser photocoagulation described above uses similar principles in the treatment of ROP. Laser treatment is directed to the anterior retina, which does not yet have blood supply. The goal of the treatment is to remove the pathological vessels before they lay enough scar tissue to cause retinal detachment. In addition, the avascular anterior retina at the edges is thinned by a laser, reducing oxygen demand and blunting angiogenic stimuli, much like cryotherapy. Laser therapy is superior to cryotherapy in that it focuses on the retina, and not on the entire thickness of the eye wall. Since laser therapy involves less tissue and is painless, inflammation significantly decreases after treatment. Compared to cryotherapy, laser therapy has advantages because there is a reduced need for anesthetics.

If laser therapy or cryotherapy is unsuccessful in stopping the progression of ROP, there are some types of surgical treatments. If there is a shallow retinal detachment due to a slight traction on the side of the fibrovascular scar tissue, a procedure called scleral flexion may be beneficial. Bending the sclera involves placing silicone tape around the equator of the eye and tightening it to create a slight indentation on the inside of the eye. This tape reduces the traction of the vitreous gel, which pulls off the fibrous scar tissue and the retina. This allows the retina to flatten onto the wall of the eye and restore normal function. Infants who have been bent by the sclera can maintain good vision, especially if there was no exfoliation of the macula. Usually, the surrounding tape needs to be removed after a few months, because the eye will continue to grow, creating a gradually increasing squeezing of the eyeball and caused nearsightedness.

In the late stage of ROP with complete retinal detachment due to scar tissue on the retina, flexion of the sclera is not enough to reduce traction. For these babies, vitrectomy can be considered. Vitrectomy involves making several small incisions in the eye and, using a suction / cutting device, removing the vitreous gel. The vitreous body is replaced with saline to maintain the shape of the eye, and the eye is able to maintain its shape and pressure indefinitely without a vitreous gel. After the vitreous has been removed, the scar tissue on the retina can be peeled off or cut off, allowing the retina to relax and fit back onto the wall of the eye. It may take several weeks for the retina to become reattached after surgery, and if holes or tears form in the retina during the procedure, the retina usually does not reattach. To allow complete excision of the scar tissue, it is often necessary to remove the lens of the eye, but some newer techniques that can preserve the lens are currently being tested.

However, the success rate of vitrectomy surgery for ROP is somewhat limited. Published data on the incidence of anatomical success, which means getting a retina attached to the wall of the eye, ranges from 25% to 50% of patients undergoing surgery. The frequency of functional success, which means the ability to see well, is significantly lower. Of the eyes that have undergone a “successful” vitrectomy operation (anatomical success), only about 1/4 are able to see well enough to reach and grab an object or distinguish between samples.

Another disabling eye disease is found in patients who have diabetes. About 14 million Americans have diabetes. In addition to numerous systemic complications (such as kidney failure, hypertension, and cardiovascular disease), diabetes is one of the leading causes of blindness among working-age Americans. Indeed, the risk of blindness in people with diabetes is 25 times higher than the risk of the general population. In many patients with diabetic eye problems, they are asymptomatic, despite the presence of a disease that threatens vision. If a diabetic eye disease is left untreated, it can lead to serious loss of vision. Diminished vision due to diabetes can be caused by several mechanisms, and treatment must be selected in accordance with 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.gov/health/diseases.htm)

Many people with diabetes notice blurred vision when their blood sugar is especially high or low. Specified blurred vision occurs as a result of changes in the shape of the lenses of the eyes and is usually eliminated when their blood sugar returns to normal levels. Diabetes mellitus is a disease that affects not only blood sugar levels, but also blood vessels. Symptoms associated with diabetes mellitus (including high blood pressure) cause damage to the microcirculatory system, including the capillaries associated with the retina. Damage to the capillaries leads to a reduced blood flow to certain areas of the retina. In addition, damaged blood vessels tend to leak, causing edema within the retina.

There are two main categories of diabetic eye diseases. The first category is called background diabetic retinopathy or non-proliferative retinopathy. This is essentially the earliest stage of diabetic retinopathy. This stage is characterized by damage to the small blood vessels of the retina, which leads to leakage of fluid (blood) into the retina. The most significant part of vision loss during this stage is due to fluid accumulating in the macula. This accumulation of fluid is called macular edema, and it can cause temporary or permanent low vision. The second category of diabetic retinopathy is called proliferative diabetic retinopathy. Proliferative retinopathy is the end result of diabetes-related damage to the retinal capillary bed (retinal vascular membrane itself). Damage to the choroid of the retina itself causes oxygen retention of the retina. Retinal tissue responds to its anoxic environment by producing angiogenic cytokines that stimulate neovascularization. As indicated previously, neovascularization of the retina causes bleeding in the eye, the formation of scar tissue of the retina, retinal detachment, and any of these symptoms can cause decreased vision or blindness. Diabetic patients also often suffer from glaucoma due to neovascularization, which is manifested by redness, blood vessels growing on the iris, which causes the closure of the corner.

Diabetic retinopathy can occur in type I diabetes (onset of diabetes before age 40) and type II diabetes (onset after 40 years), although it tends to occur more frequently and is more severe in type I patients. Since type II diabetes is often not diagnosed until the patient has had the disease for many years, diabetic retinopathy may be present in the type II patient at the time the diabetes is diagnosed.

The treatment of diabetic retinopathy depends on many factors, including the type and degree of retinopathy, related factors such as cataracts or vitreous hemorrhage, and the patient’s medical history. Treatment options include the same options that were discussed for ROP, namely laser photocoagulation, cryotherapy (freezing) and vitrectomy surgery. Blindness due to diabetic retinopathy can be prevented in most cases.

Intraocular cancers of any type are most rare. However, ocular tumors are extremely severe in that cancers of the choroid of the eye as a whole metastasize to and from other areas of the body. The most common primary malignant tumor of the eye, choroid of the choroid, occurs in 7 individuals per million of the total population per year - less than one tenth of the incidence of lung cancer. Retinoblastoma occurs as a childhood disease about as often as hemophilia. These two intraocular tumors are very different and are connected only by anatomical proximity. The choice of treatment for eye cancer depends on where the cancer is in the eye, how far it has spread, and the general health and age of the patient. (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 one of the 15,000-30000 live babies that are born in the United States. Retinoblastoma affects children of all races, both boys and girls.

A tumor (s) of retinoblastoma occurs in the retina, the photosensitive layer of the eye, which allows the eye to see. Retinoblastoma treatment is individualized for each patient and depends on the age of the child, the involvement of one or both eyes, and whether or not the cancer has spread to other parts of the body. If left untreated, the child may die. Retinoblastoma treatments include enucleation, external beam irradiation, radioactive plaques, laser therapy, cryotherapy, and chemical reduction.

Enucleation is the most common form of treatment for retinoblastoma. During enucleation, surgical removal of the eye is performed. This is necessary because it is the only way to completely remove the cancerous tumor. It is not possible to remove a cancerous tumor from within the eye without removing the entire eye. Although partial enucleation is possible for some other types of eye cancers, it is risky and may even contribute to the spread of cancer in patients with retinoblastoma.

When both eyes are involved, sometimes the more affected or “worse” eye is enucleated, while the other eye can be treated with one of the preserving methods of vision, such as external beam irradiation, plaque therapy, cryotherapy, laser therapy and chemical reduction, which described below.

External beam irradiation has been used since the early 1900s. as a way to save the eye (s) and vision. Retinoblastoma is sensitive to radiation, and treatment is often successful. Radiation therapy is carried out on an outpatient basis five times a week for a period of 3 to 4 weeks. Plaster forms are made to order to prevent head movement during treatment, and sometimes sedatives are prescribed before treatment.

Tumors usually become smaller (their regression occurs) and look healed after treatment with external radiation from the beam, but they rarely disappear completely. In fact, they may even become more apparent as they shrink, because the pinkish-gray tumor, volumetric formation is replaced by white calcium. Immediately after treatment, the skin may become tanned or a small plaque of hair may be lost on the back of the head as a result of the beam exit position. After external beam irradiation, long-term effects may include cataracts, radiation retinopathy (bleeding and exudation of the retina), impaired vision and suppression of the growth of the temporal bone (bones on the side of the head that do not grow normally). Irradiation can also increase the risk of a child developing other tumors outside the eye, in those children who have an abnormal gene in each cell of their bodies.

Radioactive plaques are discs of radioactive material that were developed in the 1930s to irradiate retinoblastoma. Currently, the iodine-125 isotope is used, and plaques are made to order for each child. In general, for this procedure, the child must be hospitalized, and he is subjected to two separate operations (one for the introduction of the plaque and one for its removal) for 3-7 days.

Laser therapy, sometimes referred to as photocoagulation or laser hyperthermia (which are two different methods), is a non-invasive treatment for retinoblastoma. Lasers effectively destroy smaller retinoblastoma tumors. This type of treatment is usually performed by focusing the light through the pupil onto the cancerous tumors in the eye and surrounding tissues. Recently, a new laser delivery device, called a diopex probe, has made it possible to treat cancer by targeting light through the wall of the eye rather than through the pupil. Laser treatment is performed under local or general anesthesia, usually it does not cause any postoperative pain associated with it, and does not require any postoperative medication. The laser can be used alone or in addition to external beam irradiation, plaques, or cryotherapy.

Cryotherapy can also be performed in patients with retinoblastoma. Cryotherapy is performed under local or general anesthesia and freezes smaller retinoblastoma tumors. A pen-like probe is placed on the sclera in close proximity to the tumor, and the tumor is frozen. Cryotherapy usually needs to be repeated many times to successfully destroy all cancer cells. A side effect of cryotherapy is that it causes swelling of the eyelids and eyes for 1 to 5 days; sometimes the swelling is so great that children are unable to open their eyelids for several days. To reduce swelling, eye drops or ointment are often prescribed.

Chemical reduction is chemotherapy for retinoblastoma. Chemotherapeutic agents are generally administered intravenously to the child, they pass through the blood stream and, if successfully treated, cause tumors to shrink within a few weeks. Chemotherapy with one or more drugs can be performed once, twice or more. Depending on the drug (s) and on the institution, the child can be hospitalized or not hospitalized during this process. After chemotherapy, the child is re-examined, and the remaining tumor (s) are treated with cryotherapy, a laser or a radioactive plaque. The number of treatment sessions that may be required in children reaches twenty with repeated eye examinations under anesthesia every 3 weeks.

Although retinoblastoma is rarely cured immediately, retinoblastoma can spread (metastasize) outside the eye to the brain, central nervous system (brain and spinal cord) and bones. In this case, a pediatric oncologist prescribes chemotherapy, and chemotherapeutic drugs are administered through peripheral blood vessels or into the brain for several months to several years after the initial diagnosis of metastatic disease.

Tumors other than retinoblastoma and melanoma appear in the eye, and they are often a harbinger of a disease of a different location. Choroid metastasis is the most common intraocular malignant lesion, and it may be the initial manifestation of systemic malignancy. Metastases in the choroid resemble unpigmented melanomas. On a fluorescein angiogram, they have an appearance similar to melanoma, and exhibit subtle echographic differences on the ultrasound echogram. However, metastases in the choroid of the eye grow faster and are more likely to cause large exudative retinal detachments.

In general, when a metastatic disease is detected in the eye, the prognosis for survival is unfavorable. However, as survival in patients with systemic cancerous lesions improves, successful treatment of ocular metastases plays an increasingly important role in maintaining a good quality of life.

Primary ocular lymphoma is one of the most intriguing intraocular tumors. Its connection with primary lymphoma of the central nervous system and the tendency of the tumor to proliferate in the subreticular pigmented epithelial space, where lymphoid tissue does not exist, are just two characteristic aspects of this highly aggressive lymphoma. It is known that the clinical manifestations of primary ocular lymphoma mimic benign inflammatory lesions of the choroid and, thus, delay the correct diagnosis for months. Neoplastic cells with ocular lymphoma may remain confined in the space between the retinal pigment epithelium and the Bruch membrane. Since the vitrite associated with these lymphoma aggregates often consists of reactive lymphocytes, a vitreous biopsy may not be diagnostic. This has led to the false concept that it is difficult to interpret intraocular cytology when surgeons do not actually receive tumor cells. The positive yield as a result of an intraocular biopsy can be increased in some cases if the surgeon performs an aspiration biopsy through retinotomy in the retinal pigment epithelial space. Primary ocular lymphoma consists of large, cytologically atypical cells that stain positively on the total leukocyte antigen. Aspirates are usually associated with large numbers of necrotic tissue fragments. In the past, immunophenotypic analysis was problematic. Some early studies failed to detect any surface markers, and their authors concluded that ocular lymphoma is a tumor of "null" cells. Pretreatment of cells with hyaluronidase increased the yield of immunopathological studies.

Another form of ocular cancer is choroid melanoma. Choroidal melanoma is a primary eye cancer. It comes from the pigmented cells of the choroid and does not represent a tumor that occurs elsewhere and spreads to the eye. Although some choroid melanomas are more life threatening than others, almost all of them should be treated as if they were malignant. It turns out that some choroid melanomas remain "dormant" and do not grow. Most of them increase slowly over time and lead to loss of vision. These tumors can spread to other parts of the body and ultimately lead to death. Numerous cases of ocular melanoma metastatic to the liver have been reported (Source: The Eye Cancer Network: www.eyecancer.com).

For many years, enucleation has been the usual treatment for choroidal melanoma. If the tumor has not spread to other parts of the body, removing the eye as a whole completely relieves the patient of the tumor. Beginning in World War II, radiation was used for choroidal melanoma. Over the past 20 years, this method of treatment has been improved. Irradiation in appropriate dose frequencies and in the necessary physical forms is aimed at eliminating the growing tumor cells without causing damage to the normal tissue, sufficient to require removal of the eye. As the cells die, the tumor shrinks, but it usually does not completely disappear. The most promising widely available method of irradiating middle melanoma of the choroid of the eye involves the construction of a small plaque with radioactive granules glued on one side. However, exposure is usually accompanied by adverse side effects such as vomiting and baldness.

High-energy particles (irradiation with helium ions or a proton beam) from a cyclotron can also be used to irradiate tumors. The operation is performed first to sew small metal clips to the sclera so that a particle beam can be precisely aimed. Treatment is carried out for several consecutive days. The equipment needed for these treatments is available in only a few medical centers in the world. Good results have been reported in some patients, but many patients treated in this way have only been observed for several years. Therefore, the long-term results of these forms of radiation therapy, in comparison with the more widely used plaque, are unknown.

For many years, a small number of patients have used other methods of treatment. Photocoagulation using white light or laser light was used to burn small tumors, and cryotherapy was used to kill the tumors by freezing them. It is believed that these techniques are effective only in very small tumors. Some doctors combined laser or cryotherapy with radiation, but such treatments are experimental. Several patients underwent resection of the eye wall or a related procedure to remove tumors from their eyes. These treatment methods are considered experimental by most doctors and were used only for a small number of tumors. There is no cure that can guarantee the destruction of the tumor, maintain vision, or provide a normal life expectancy.

Another eye cancer is intraocular melanoma, a rare type of cancer in which cancer cells are found in a part of the eye called the choroid of the eyeball. The choroid of the eyeball contains cells called melanocytes, which contain pigment. When these cells become malignant, the cancer is called melanoma. The vascular membrane of the eyeball includes the iris (colored part of the eye), the ciliary body (the muscle in the eye) and the choroid itself (the layer of tissue in the back of the eye). The iris opens and closes to change the amount of light entering the eye. The ciliary body changes the shape of the lens inside the eye so that it can focus. The choroid itself is located after the retina, the part of the eye that creates the picture. If there is melanoma that begins in the iris, it may look like a dark spot on the iris. If melanoma is located in the ciliary body or the choroid of the eye itself, the person may have blurred vision or no symptoms, and the cancer may grow until it is noticed. (Source: The Eye Cancer Network: www.eyecancer.com).

The likelihood of recovery (prognosis) for intraocular melanoma depends on the size and cell type of the cancer, where the cancer is located in the eye, and whether the cancer has spread. There are treatments for all patients with intraocular melanoma. Three types of treatment are usually prescribed, namely surgery (removal of a cancerous tumor), radiation therapy (using a high dose of X-rays or other high-energy rays to "destroy" the cancer cells) and photocoagulation (destruction of the blood vessels that feed the tumor).

Surgery is the most common treatment for intraocular melanoma. A doctor can remove a cancerous tumor using one of the following operations:

- iridectomy - removal of only parts of the iris, and;

- iridotrabeculectomy - removal of parts of the iris and supporting tissues around the cornea, a transparent layer covering the front of the eye;

- iridocyclelectomy - removal of parts of the iris and ciliary body;

- choroidectomy - removal of parts of the choroid proper;

- enucleation - removal of the entire eye.

Radiation therapy can also be used to deliver X-rays or other high-energy rays to the area where the cancer cells exist, in order to destroy the cancer cells and shrink the tumor. Irradiation can be used alone or in combination with surgery. You can also use photocoagulation treatment, in which a very small beam of light rays, usually from a laser, is applied to the eye to destroy blood vessels and destroy the tumor.

The vast majority of the proposed methods of treatment for the treatment of eye diseases, in particular, subcellular neovascularization and eye tumors, initially use surgery or radiation therapy. In the treatment of patients with drugs, separately or after surgery, the introduction of the drug as a whole is carried out systemically, or by injection, or orally. As noted above, both surgery and radiation therapy for eye diseases are painful, often require long recovery periods, and may be accompanied by adverse side effects. In addition, systemic administration by ingestion of a drug or injection into a place other than the eye is often provided in ineffective amounts, necessitating the administration of often unacceptably high levels of the drug to achieve effective intraocular concentrations. Thus, there is a great need for successful non-systemic therapy for the treatment of ocular diseases, such as neovascularization in the cornea and retina. In addition, drug and drug delivery to the eye without adverse side effects remains a major problem. The invention under discussion provides such therapy, providing effective non-systemic administration of a tubulin binding agent for the treatment of ocular disease with minimal side effects.

SUMMARY OF THE INVENTION

The present invention is directed to the administration of a vascular-targeted agent ("VTA"), in particular a tubulin binding agent, for the treatment of malignant or non-malignant vascular proliferative disorders in ocular tissue.

Neovascularization of ocular tissue is a pathological condition characterized by vascular proliferation, and it occurs in various vascular diseases with varying degrees of visual impairment. The introduction of VTA for the pharmacological control of neovascularization associated with non-malignant vascular proliferative disorders, such as wet macular degeneration, proliferative diabetic retinopathy or retinopathy of premature infants, would potentially be beneficial for patients for whom there are few treatment options. In another embodiment, the invention provides the administration of VTA to pharmacologically combat neovascularization associated with malignant vascular proliferative disorders, such as ocular tumors.

The blood-brain barrier (BRB) is composed of specialized unfenestrated tightly connected endothelial cells that form a transport barrier for certain substances between the retinal capillaries and retinal tissue. The arising vessels of the cornea and retina associated with retinopathies are aberrant, very similar to those associated with solid tumors. Tubulin binding agents, tubulin polymerization inhibitors, and vascular targeting agents may be able to attack the resulting aberrant vessels, because these vessels do not have common architectural analogies with the blood-brain barrier. Tubulin-binding agents can stop the progression of the disease very much like how they enter the tumor vasculature. Local (non-systemic) delivery of tubulin-binding agents can be achieved using injections into the vitreous humor, injections under the episcleral space, iontophoresis of eye drops and implants and / or inserts. Systemic administration can be accomplished by introducing tubulin binding agents into the blood stream to a place that is separated by a measurable distance from a diseased or affected organ or tissue, in this case the eye. Preferred methods of systemic administration include parenteral or oral administration.

Details of one or more embodiments of the invention are set forth below. Although any methods and materials similar or equivalent to those described can be used in the practice or testing of the present invention, preferred methods and materials are disclosed herein. Other features, objects, and advantages of the invention will be apparent from the description. In the description and the attached claims, the singular also includes the plural unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as the meaning commonly understood by any person skilled in the art for whom this invention is intended. All patents and publications cited in this specification are incorporated herein by reference.

Detailed Description of Drawings

The invention will be better understood with reference to the accompanying drawings, in which:

Figure 1 is a simplified anatomical illustration of a mammalian eye in front and side;

Fig. 2A illustrates a healthy macula; Fig. 2B illustrates the dry form of macular degeneration;

Fig. 2C illustrates a wet form of macular degeneration;

3A and 3B are enlarged photographs of a portion of the cornea showing inhibition of vascular growth on the 28th day after administration of CA4P, in comparison with the control eye after administration of the carrier;

Figa and 4B illustrate the microscopic histology of changes in the cornea (inhibition of vascular growth) on the 28th day after administration of CA4P, compared with the control eye after administration of the carrier.

Fig. 5A illustrates the effect of a single dose of CA4P on vascularization of an ocular tumor in an animal model of retinoblastoma;

5B illustrates the degree of tumor regression in an experimental model of retinoblastoma in an animal after repeated administration of CA4P.

DETAILED DESCRIPTION OF THE INVENTION

The human eye has several structurally unique properties: it is exposed to the environment, it is richly equipped with nerves, it has a high blood flow velocity in the choroid of the eye itself, while the anterior chamber and vitreous body are completely avascular and isolated from the circulatory system. The exceptional eye architecture provides sufficient opportunity for the delivery of tubulin binding agents by one or more non-systemic routes of administration for the treatment of ocular conditions, diseases, tumors and disorders. A simplified anatomical illustration of the eye is shown in figure 1.

As indicated earlier, neovascularization of ocular tissue is a pathogenic condition that occurs with various eye diseases and is associated with varying degrees of visual impairment. A pharmacological fight against neovascularization would be potentially beneficial for patients suffering from diseases such as wet macular degeneration, proliferative diabetic retinopathy, and premature retinopathy.

Tubulin binding agents inhibit tubulin assembly by binding to tubulin binding cofactors or cofactor-tubulin complexes in the cell during mitosis and prevent cell division and, consequently, cell proliferation. Tubulin binding agents include a wide class of compounds that inhibit tubulin polymerization and which generally function as tumor selective vascular targeting agents that can be used for cancer chemotherapy as well as for other uses other than cancer treatment, such as treating eye diseases.

As discussed above, one of the drawbacks of systemic administration of drugs for treating eye diseases is that systemic administration as a whole does not provide effective levels of the drug specifically in the eye. Since systemically administered drugs can be metabolized in the body even before they reach the eye, high levels of the drug will be required to achieve effective intraocular concentrations. Unsystematic or local injection of drugs directly into the eye (s) of a patient suffering from an eye disease provides an opportunity to achieve an effective concentration of the drug to be administered, and is very favorable for the patient.

Ocular indications that can be treated by the non-systemic administration of tubulin binding agents in accordance with the present invention include non-malignant vascular proliferative diseases characterized by neovascularization of the cornea, retina or the choroid itself, as well as malignant vascular proliferative diseases such as ocular tumors and cancerous lesions. Corneal neovascularization occurs in the following diseases: trachoma (Chlamydia trachomatis), viral interstitial keratitis, microbial keratoconjunctivitis, transplantation and burns of the cornea. It can be caused by infection (trachoma, herpes, leishmaniasis, onchocerciasis), transplantation, burns (thermal, alkaline), trauma, malnutrition and damage caused by a contact lens. Neovascularization of the retina and / or the choroid itself occurs with macular degeneration, diabetic retinopathy, and premature retinopathy. Neovascularization of the anterior chamber occurs with glaucoma.

Non-systemic methods of administering tubulin binding agents provided by the present invention include: administration to the vitreous body (injection), administration under the conjunctiva, administration to tissues surrounding the eye, injection into the episcleral space, iontophoretic delivery, topical administration by eye drops, gels or ointments and through an eye liner or implant.

Tubulin binding agents can be administered to the vitreous by injection directly into the vitreous of the eye. Tubulin binding agents can also be administered under the conjunctiva by injection under the conjunctiva and around the eye by injection into the tissues surrounding the eye.

Tubulin binding agents can also be injected into the episcleral space (under the Tenon capsule) with a blunt-tipped Connor cannula. Using the appropriate technique, a healthcare provider administering a dose of tubulin binding agent can avoid puncture the eyeball and damage to the optic nerve. After delivery, the injection site is cauterized, and the space serves as a depot of the drug. Introduction to the episcleral space is less invasive than injection into the vitreous.

In another embodiment of the present invention, the tubulin binding agent may be formulated as a biocompatible, biodegradable and / or biologically erodible implant or insert containing a tubulin binding agent in order to provide a slow release of the drug and maintain a therapeutically effective concentration of the drug in for a long period of time. Biologically erodable ocular implants containing a drug for implantation or insertion into a mammalian eye are described, for example, in US Pat. No. 5,904,144 and US Pat. No. 5,766,242, which are incorporated herein by reference in their entirety. Ophthalmic implants generally include a capsule that is placed in a desired position in the eye. The capsule may include one or more drugs or may include cells that produce a biologically active molecule for sustained, controlled delivery to the eye. The amount of drug that can be used in this embodiment will vary depending on the effective dosage of the drug and the rate of release from the liner or implant to the eye or into it.

Due to the fact that the sclera is exposed, an iontophoretic probe can be placed on the surface of the eye. In iontophoresis, an electric current is used to stimulate the flow of ionic compounds through the cell membrane. This technique is currently used for transdermal delivery of ionic preparations. The two main mechanisms by which iontophoresis stimulates drug transport are: (a) iontophoresis, in which a charged ion is displaced from an electrode of the same charge, and (b) electroosmosis, the convective movement of a solvent that occurs through a charged “pore” in response to a preferred passage counterions when an electric field is applied.

The tubulin binding compositions may also be formulated for topical administration to the eye in the form of sterile, eye drops.

In accordance with the present invention, the preferred tubulin binding agent is combretastatin A4 (“CA4”), a powerful vascular-targeting agent. CA4 is essentially insoluble in water. This characteristic interferes with the formulation of pharmaceutical compositions of this compound. Thus, the most preferred prodrug form of combretastatin A4 (“CA4P”) is used to compensate for the generally poor solubility of CA4. However, the invention is not limited in this regard, and CA4 compositions may work just as well or better than CA4P.

Combretastatins are obtained from tropical and subtropical shrubs and trees of the Combretaceae family, which represent a practically unexplored reservoir of new substances with potentially useful biological properties. Illustrative is the genus Combretum with 25 species (10% of the total), known in primitive medical practices in Africa and India for such diverse applications as the treatment of leprosy (See: Watt, JM et al, "The Medicinal and Poisonous Plants of Southern and Eastern Africa ", E. & S. Livingstone, Ltd., London, 1962, p. 194) (the root of the species Combretum) and cancer (Combretum latifolium).

It was found that combretastatins are antitumor substances. Numerous combretastatins were isolated, structurally investigated and synthesized. US Patent Nos. 5,409,995 and 5,597,886 describe the isolation and synthesis of combretastatins designated A-1, A-2, A-3, B-1, B-2, B-3, and B-4. Descriptions of these patents are fully incorporated into this description by reference. A related combretastatin, designated “Combretastatin A4,” has been described in US Pat. No. 4,996,237 to Petit, which is incorporated herein by reference in its entirety.

CA4P is a derivative of the naturally occurring combretastatin A4 subtype described in US Pat. No. 5,561,122, the entire disclosure of which is incorporated herein by reference. The preferred CA4P compound replaces the disodium phosphate derivative with an —OH group in the CA4 structure, and this compound allows the reverse metabolic conversion of CA4P to water-insoluble CA4 in vivo. However, the invention is not limited to the phosphate derivative, and other prodrug moieties can be substituted for the —OH group in compound CA4. In addition, it is expected that phosphate salts of prodrugs other than the disodium salt of CA4P work essentially the same way for the purposes of this invention. Examples of other phosphate salts of prodrugs are described in PCT patent applications WO 02/22626 and WO 99/35150, the descriptions of which are included in the present description.

CA4P is the first in a new class of drugs — antitumor vascular-targeting drugs — that cause solid tumors to shrink in volume by selectively targeting and destroying tumor-specific blood vessels formed by angiogenesis. Targeted action against tumor vessels and inhibition of angiogenesis are related cancer treatments that radically depart from conventional cancer treatment approaches. Unlike traditional methods, including a direct attack on cancer cells, these new drugs are aimed at maintaining the life of the tumor, a network of newly emerging blood vessels that form as a result of angiogenesis, the germination of new blood vessels from previously existing vessels. Preclinical studies have shown that the use of these methods of treatment can cause a reduction in volume and ultimately the disappearance of the tumor. In addition, when CA4P was used on in vitro and in vivo animal cell models, it showed unusual vascular toxicity (Int. J. Radiat. Oncol. Biol. Phys. 42 (4): 895-903, 1998; Cancer Res. 57 (10): 1839-1834, 1997).

Although both angiogenesis inhibitors and anti-tumor vascular agents, such as combretastatins, target the blood vessels of the tumor, they differ in their approach and outcome. When inhibiting angiogenesis, the goal is to prevent tumor growth by inhibiting the formation of tumor-specific blood vessels that nourish and support the tumor. On the other hand, when targeting against tumor vessels, the goal is to obliterate the tumors by selective exposure and destruction of their existing blood vessels, creating a quick and irreversible shutdown of these blood vessels. This effect is not observed when using drugs against angiogenesis. Only antivascular targeting activity can destroy existing blood vessels that support tumor growth. Combretastatin is also able to inhibit the proliferation of endothelial cells that produce and line the new vasculature of the tumor (antiangiogenic activity). Therefore, it is believed that combretastatin can behave both as a drug targeting tumor vessels and as an anti-angiogenic drug. Preclinical studies have shown that both treatments leave the blood vessels associated with healthy tissue unaffected. The present invention provides for the administration of CA4P, both alone and / or in combination with modern drugs for the treatment of eye diseases.

A vasculature formed by angiogenesis has also been observed in diseases other than cancer, including eye diseases such as macular degeneration, proliferative diabetic retinopathy, and neonatal retinopathy. Preliminary work to reduce such a vasculature in an experimental model of the eye was conducted in the laboratory of Donald Armstrong, Ph.D., D.Sc., University of Florida, College of Veterinary Medicine, Division of Ophthalmology, which showed that CA4P accelerated the rate of reverse development pre-formed vessels in the eye of an experimental animal model. 3A, 3B, 4A and 4B show the reverse development of preformed vessels in the eyes of rabbits examined in this experiment.

CA4 and CA4P are currently undergoing clinical trials for the treatment of a variety of diseases and indications, including use as agents targeting tumor vessels and as an inhibitor of angiogenesis. In addition, CA4P has demonstrated the ability to treat ocular diseases such as reticulum neovascularization. The present invention also provides the use of synthetic analogues of combretastatins, as described in the publication Bioorg. Med. Chem. Lett. 11 (2001) 871-874, 3073-3076, J. Med. Chem. (2002), 45: 1697-1711, WO 02/50007, WO 01/12579, WO 00/35865, WO 00/48590, WO 01/12579, US patent No. 5525632, US patent No. 5674906 and US patent No. 5731353.

Other tubulin binding agents that can be administered as VTA include the following agents or their prodrugs: 2,3-disubstituted benzo [b] thiophenes (US Patent Nos. 5886025; 6162930 and 6350777), 2,3-disubstituted benzo [b ] furans (WO 98/39323), 2-3-disubstituted indoles (WO 01/19794), disubstituted dihydronaphthalenes (WO 01/68654) or analogs of colchicine (WO 99/02166). In addition, additional non-cytotoxic prodrugs of vascular targeting agents that are converted substantially into a cytotoxic drug by the action of an endothelial enzyme selectively inducible at elevated levels in vascular proliferation sites, as described in WO 00/48606.

Additional known tubulin inhibiting agents that can be administered in accordance with the present invention include: taxanes, vinblastine (vinca alkaloids), colchicines (colchicinoids), dolastatins, podophyllotoxins, steganacins, amphtetinyls, flavanoids, rhizoxins, curacins A, efotilones A and curatins A , velvistatins, phenstatins, 2-styrylquinazolin-4 (3H) -ones, stilbenes, 2-aryl-1, 8-naphthyridin-4 (1H) -ones and 5,6-dihydroindole (2,1-a) isoquinolines.

Regarding the administration and delivery of tubulin-binding agents to the eye of a subject in need, it is important to consider that the human eye has several structurally unique properties: it is exposed to the environment, it is richly equipped with nerves, it has a high blood flow velocity in the choroid itself eyes, while the anterior chamber and vitreous body are completely avascular and isolated from the circulatory system. The exceptional eye architecture provides ample opportunity for alternative drug delivery methods. In this regard, four non-systemic routes of administration are provided by the present invention, namely, intravitreal injection (injection), injection into the episcleral space, iontophoretic delivery, implants / inserts, and eye drop delivery.

The results of eye irritation and studies of the biological distribution and inhibition of vascular growth in experimental models of neovascularization of the cornea in animals, the choroid of the eye or retina after administration of CA4P are described below in the Examples section.

Neovascular retinopathies, as well as ocular tumors, are thus a viable target for CA4P therapy and other tubulin binding agents for a variety of reasons, namely:

- Tubulin binding agents may be able to attack the resulting aberrant vessels associated with ratinopathy, because these vessels do not have common architectural analogies with the blood-brain barrier (BRB). Tubulin-binding agents can stop the progression of the disease very much like how they enter the tumor vasculature. In addition, tubulin binding agents may be able to cause regression of developing vessels, as has been observed in various preclinical studies.

- Since there are no 100% effective treatments for subcellular neovascularization, tubulin-binding agents can be effective when used in combination with treatment methods in the current state of the problem.

- Most currently approved treatments for retinopathies include surgery, which can be painful and require long recovery periods. Non-systemic or systemic administration of tubulin binding agents may be a non-surgical treatment.

- With systemic and non-systemic delivery, CA4P is promising as a vascular-targeting agent in experimental models of angiogenesis of the cornea, retina, or choroid proper, and in experimental models of ocular tumors.

As indicated, CA4P, as well as other vascular-targeting and tubulin-binding agents, are promising for systemic delivery of models of angiogenesis of the cornea, retina, or choroid proper, as well as other eye diseases and tumors. Preferred methods of systemic administration include parenteral and oral administration. Parenteral administration is the route of administration of drugs by injection under or through one or more layers of the skin or mucous membranes. Parenteral routes of administration, by definition, include any route other than the oral-gastrointestinal (enteral) tract. Parenteral administration includes intravenous, intramuscular and subcutaneous routes.

The pharmaceutical compositions of the invention are formulated for compatibility with the intended route of administration. Pharmaceutical compositions for topical topical administration may include ophthalmic solutions, ophthalmic gels, aerosols, ointments, perfusion solutions and inserts. A locally delivered tubulin binding agent composition must remain stable for a period of time long enough to achieve the desired therapeutic effects. In addition, the agent must penetrate through the surface structures of the eye and accumulate in significant quantities in the area of the disease. In addition, a locally delivered agent should not cause an excessive amount of local toxicity.

Ophthalmic solutions in the form of eye drops generally consist of aqueous media. In order to meet a wide range of drugs that have varying degrees of polarity, buffers, organic carriers, inorganic carriers, emulsifiers, wetting agents, etc. can be added. Pharmaceutically acceptable buffers for topical ophthalmic compositions, among others, include phosphate, borate, acetate and glucuronate buffers. Drug carriers may include water, an aqueous mixture of lower alkanols, vegetable oils, polyalkylene glycols, liquid paraffin, ethyl cellulose, ethyl oleate, carboxymethyl cellulose, polyvinyl pyrrolidone and isopropyl myristate. Ophthalmic aerosols generally give the same results as eye drops and can be formulated in a similar manner. Some ophthalmic preparations have poor penetration through the eye barriers and cannot be administered as drops or aerosol. Thus, ointments can be used to extend contact time and increase the amount of absorbed drug. Long-term and permanent perfusion of the eye with solutions containing the drug can be achieved by placing a plastic tube in the conjunctival sac. The flow rate of the perfusate can be adjusted by means of a mini-pump to create continuous irrigation of the eye. The inserts are similar to soft contact lenses located on the cornea, except that the inserts are generally placed in the upper blind sac or, less commonly, in the lower conjunctival sac, rather than attached to the open cornea. The liners are generally made from biologically soluble materials that dissolve in the tear fluid or disintegrate while simultaneously releasing the drug.

In one embodiment, the active compounds are applied to implants or inserts that are implanted in the eye. One example of such an implant provided by the present invention is an implant from Oculex Pharnaceuticals, Inc., Sunnyvale, CA. The Oculex implant is a BDD ™ biodegradable drug delivery device consisting of a biodegradable microscopic polymer system that allows microencapsulated therapeutic systems to be implanted into the eye. The implant provides the ability to release the desired drug directly into the eye area, requiring drug exposure for a given period of time from several days to several months, and even up to periods reaching many years.

It is particularly preferable to formulate the local compositions in a dosage form for ease of administration and uniformity of dosage. As used herein, the term "dosage form" refers to physically discrete units selected as unit dosages for the subject to be treated, each unit containing a predetermined amount of active compound, calculated to cause the desired therapeutic effect, in connection with the desired pharmaceutical carrier . The specifications of the dosage forms of the invention are dictated and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and on the limitations inherent in the problem of mixing such an active compound for treating individuals. Further known information regarding the methods for making the compositions of the present invention can be found in standard references in the art, such as, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easter, PA, 15 ^th Ed. (1975). In addition to the non-systemic routes of administration discussed previously, examples of systemic routes of administration include parenteral, for example, intravenous, intradermal, subcutaneous, oral (e.g., inhalation) administration, mucosal administration, and rectal administration. Solutions or suspensions used for parenteral or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be placed in ampoules, disposable syringes or bottles made of glass or plastic, containing many doses.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions, and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily inserted into the syringe. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerin, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the case of dispersion, and using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the compositions for injection can be achieved by the inclusion in the composition of an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a vascular targeting agent) in the required amount in an appropriate solvent, as required, with one or a combination of the above ingredients, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and lyophilization, which gives the powder of the active ingredient plus any additional desired ingredient from its pre-sterilized solution by filtration.

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 may be included with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouth rinse, the compound in a fluid carrier being introduced into the oral cavity and spilled or swallowed after rinsing. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds of a similar nature: a binder, such as microcrystalline cellulose, tragacanth resin or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glossing agent such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or an orange flavor.

For administration by inhalation, the compounds are delivered by aerosol spraying from a pressurized container or delivery means that contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be via administration through the mucous membranes or through the skin. For administration through the mucous membranes or through the skin, substances are used in the composition to promote penetration through the corresponding barrier through which they must penetrate. Such penetration enhancers are generally known in the art and include, for example, mucosal administration of detergents, bile salts, and fusidic acid derivatives. The introduction through the mucous membranes can be accomplished through the use of aerosols for the nasal cavity or suppositories. For transdermal administration, the active ingredients are included in various ointments, gels, or creams, as is generally known in the art.

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

In addition to the tubulin binding agents described above, the invention also includes the use of pharmaceutical compositions and formulations comprising the tubulin binding agent in association with, but not limited to, a pharmaceutically acceptable carrier, diluent or excipient, such as, for example, water, glucose , lactose, hydroxypropyl methylcellulose, as well as other pharmaceutically acceptable carriers, diluents, or excipients generally known in the art.

Used the terms "pharmacologically effective amount", "pharmaceutically effective dosage" or "therapeutically effective amount" means the amount of the drug or pharmaceutical agent that will cause a biological or medical reaction of the tissue, system, animal or person that the researcher or clinician expects. The dosage of CA4P for administration to the subject's eye (if administered intermittently) is in the range of about 0.01 mg / ml to about 100 mg / ml. The concentration of CA4P achieved in the eye should be therapeutically relevant and should be in the range of about 1 nanomole to 100 millimoles. A more preferred concentration of CA4P in the eye is in the range of about 1 micromole to 100 micromole. When CA4P is administered systemically, the amount of combretastatin A4 prodrug in the range of about 0.1 mg / m ² to about 120 mg / m ^{2 is} preferably administered parenterally.

It is contemplated that systemic and non-systemic administration of tubulin binding agents in accordance with the present invention will be intended for administration to mammals, in particular humans. However, the invention is not limited in this regard, and formulations may also be prepared in accordance with veterinary guidelines for administration to animals.

The invention is further defined by reference to the following examples. For specialists in this field it will be obvious that many modifications and materials, and methods can be carried out without departing from the purpose and interest of the invention.

EXAMPLES

Example 1. Eye irritation studies and determination of the average tolerated dosage (SPD) of CA4P when administered topically to the eye using three routes of administration

(i) Introduction to the vitreous body

The test article, CA4P, was evaluated to determine the possibility of causing intraocular irritation after injection into the vitreous in rabbits. After general anesthesia, CA4P at a dose of 0.2 ml was injected into the right eyes of eight rabbits. A 0.9% sodium chloride solution in accordance with the US Pharmacopoeia at a dose of 0.2 ml was injected into the left eyes of rabbits so that they served as negative controls. 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 injected into the right eyes of two rabbits. About 48 hours after treatment, the eyes were examined with a biomicroscopic slit lamp and an indirect ophthalmoscope. Scores were recorded and rabbits euthanized. Immediately after euthanasia, samples of the vitreous fluid were aspirated, and the number of leukocytes was determined using a hemacytometer. Amounts less than or equal to 200 cells / mm ³ were considered acceptable.

In the control eyes, there were no significant changes in the eye tissues based on a biomicroscopic slit lamp and an ophthalmoscope. In the eyes subjected to the test treatment, these irritations were observed in one animal at a concentration of 1 mg / ml and in both at a concentration of 100 mg / ml. The average number of cells in the vitreous fluid was 9 cells / mm ³ for the eyes, which were injected with a concentration of 0.1 mg / ml; 962 cells / mm ³ for the eyes, which were injected with a concentration of 1 mg / ml; 10 cells / mm ³ for the eyes, which were injected with a concentration of 10 mg / ml; 409 cells / mm ³ for eyes, which were injected with a concentration of 100 mg / ml and 5 cells / mm ³ for control eyes.

Under the conditions of the study, the controls responded, as expected, with the absence of a significant reaction noted in eye studies and vitreous fluid analysis. The animals subjected to the test treatment, both animals, which were injected with a concentration of 100 mg / ml, had severe irritation and inflammation in both ophthalmic studies, and they showed evidence of inflammation by analysis of leukocytes in the vitreous fluid. For all lower dose levels (0.1 mg / ml, 1.0 mg / ml and 10 mg / ml), there was no clear evidence of irritation or inflammation.

(ii) Introduction to the subepiscleral space

The test article, CA4P, was evaluated to detect primary eye irritation. Two injections in a volume of 0.1 ml of an appropriate concentration of CA4P (0.1, 1.0, 10, and 100 mg / ml) were injected into the subepiscleral space of the right eye of two rabbits. A portion in a volume of 0.2 ml of saline with buffer was injected into the subepiscleral space of the left eyes so that they served as a negative control. Ocular reactions were evaluated 24, 48, and 72 hours after sample administration. On day 3, rabbits were euthanized and their eyes removed. Samples were fixed and poured and histological examination was performed. Pathological changes in the tissues of the eye were recorded with particular attention paid to the study of changes in the subepiscleral space.

In the conditions of this study, no irritation was observed in the eyes treated with concentrations of 0.1 mg / ml and 1.0 mg / ml, compared with the corresponding eyes, which served as negative controls. Slight irritation was observed in the eyes treated with concentrations of 10 mg / ml and 100 mg / ml, compared with the corresponding eyes, which served as negative controls.

(iii) Local drops

The test article, CA4P, was evaluated to detect primary eye irritation. A single dose of 0.2 ml of CA4P dilution (0.1, 1.0, 10 and 100 mg / ml) was placed in the lower conjunctival sac of the left eye in order to serve as a comparative control. Buffered saline was injected into the opposite eye. Ocular reactions were evaluated 24, 48, and 72 hours after sample administration.

Under the conditions of this study, the macroscopic reaction to all dilutions of the test product was considered insignificant compared to control reactions. Microscopically tested product was not regarded as irritating, compared with the control product in the form of a saline solution with a buffer.

A summary of the results is presented in the table:

Draize scores Introduction Irritation* Local 0-10 mg No annoyance Vitreous <1 mg No annoyance 10 mg Irritation Subepiscleral 0-10 mg No annoyance * Dose range: 0.001, 0.01, 0.1, 1.0, 10 mg

Example 2. Evaluation of the biological distribution of CA4P at local injection into the eye using different routes of administration

In order to be effective in the treatment of ocular neovascularization, an unsystematic method of administering the drug must ensure its penetration through relevant structures in the eye and the delivery of the drug in therapeutically significant amounts to the site of the disease. To confirm that various methods of non-systemic injection can lead to a significant biological distribution of CA4P, experiments were conducted on the biological distribution of a drug labeled with a radioactive label.

Methods

Unless noted below, the following protocol was followed for each biological distribution experiment.

¹⁴ C-CA4P (OXiGENE Inc., Watertown, MA) was resuspended in 100 μl of saline and injected with a 30g needle into the right eyes of anesthetized male New Zealand rabbits (4 months old, weighing 1.8-2.5 kg, n = 3 per sample). Three different concentrations of ¹⁴ C-CA4P (1, 10, 100 mg / ml) were tested, corresponding to doses of 1, 5 and 50 μCi of exposure to radioactivity, respectively. An untreated control group was also included. Rabbits were anesthetized after 1, 6, 24, and 48 hours to collect blood samples. After blood sampling, the animals were euthanized by phenobarbital injection and the treated eyes were removed from the test animals. Samples of eye tissue were cut off from the cornea, aqueous humor, vitreous fluid, the choroid itself or the retina, placed in 20 ml glass scintillation vials, mixed by shaking, and incubated for 24 hours with 500 μl of digesting fluid. Plasma was separated from whole blood by centrifugation (1800 g for 10 minutes). Ophthalmic tissue and plasma samples were incubated at room temperature with 16 ml of Hionic Fluor scintillation fluid (for a period of 24 hours before counting the radioactivity. Each sample was counted for 5 minutes on a Betamatic V counter (Bio-Tek Kontron Instruments, St Quentin en Yvelines, France ). The conversion of cycles per minute ("cpm") to decays per minute ("dpm") was carried out automatically by a beta counter using calibration curves obtained from ¹⁴ C standards and blanking curves from the corresponding blank matrices subjected to injections of ¹⁴ C standards. Concentration med Tween funds were determined in accordance with the nanogram equivalents of CA4P (ng-Equ / g of tissue), which was calculated by the measured dpm, the weight of the tissue sample and the specific activity of the drug (0.37 mCi / mg), followed by subtraction of the corresponding background value from the control eye tissue Tissue concentration of 1 μmol CA4P is 440 nEq / g of tissue.

Results:

(i) Introduction to the vitreous body

Table 1 shows the results of the biological distribution after injection into the vitreous body. In all tissues examined, the degree of penetration into the eye depended on the concentration of CA4P placed on the surface of the eye. The highest concentrations of the drug in the eye ("C _max ") were achieved within the first hour after administration. Therapeutically relevant drug concentrations (> 1 μmol) were delivered to the retina at all concentrations studied. High concentrations of the drug were also found in the vitreous and cornea. Relatively little drug was found in the aqueous humor of the eye or blood plasma.

Table 1
Biological distribution of CA4P after injection into the vitreous body Eye tissue sample Dose (mg / ml) C _max (μmol) Watery moisture one hundred 460 10 0.79 one 0.15 Vitreous body one hundred 10,039 10 757 one 75 Retina one hundred 10,696 10 1,981 one 160 Cornea one hundred 5,871 10 969 one 95 Sclera one hundred 16,112 10 153 one 19 Plasma one hundred 0.78 10 0.2 one 0.4

(ii) Introduction to the episcleral space

(1) Biological distribution

Table 2 shows the results of a study of the biological distribution after injection into the subepiscleral space. In all the tissues examined, the degree of penetration into the eye depended on the concentration of CA4P introduced into the subepiscleral space. The highest concentrations of the drug in the eye were achieved within the first hour after administration. Therapeutically relevant concentrations of the drug (> 1 μmol) were delivered to the retina and the choroid proper at an administered dose of 100 and 10 mg / ml. High concentrations of the drug were also found in sclera. Relatively little drug was found in the vitreous, aqueous humor of the eye, or blood plasma.

table 2
Biological distribution of CA4P after injection into the subepiscleral space Eye tissue sample Dose (mg / ml) C _max (μmol) Watery moisture one hundred 5.92 10 1.8 one 0.3 Vitreous body one hundred 3.9 10 0.13 one 0.019 Retina one hundred 171 10 7.1 one 0.85 Actually choroid one hundred 861 10 6.2 one 1,2 Plasma one hundred 9.3 10 0.49 one 0,07

(iii) Subconjunctival administration

Subconjunctival injections were administered at doses of 0.1, 1, 10 and 100 mg / ml. Solutions of ¹⁴ C-CA4P were in the composition with 0.24% 10 N. KOH and 0.01% benzalkonium chloride and were injected in a volume of 100 μl. Animals were treated with an applied dose of 5 μCi by a single subconjunctival injection into the right eye. After delivery, the eyes were neatly kept closed for 2-5 seconds. The experimental results are presented below in table 3.

The highest concentrations of the drug in the eye were observed within the first hour after administration. Therapeutically relevant concentrations of the drug (> 1 μmol) were delivered to the cornea, retina and choroid proper at a dose of 100, 10 and 1 mg / ml. Relatively little drug was found in the vitreous or plasma.

Table 3
Biological distribution of CA4P after subconjunctival injection Eye tissue sample Dose (mg / ml) C _max (μmol) Cornea one hundred 761 10 143 one 13 Vitreous body one hundred 15.7 10 2.1 one 0.02 0.1 0.005 Retina one hundred 174 10 39.6 one 1.7 0.1 0.13 Actually choroid one hundred 908 10 188 one 1.9 0.1 2,3 Plasma one hundred 8 10 0.9 one 0,025 0.1 0.017

(iv) Introduction to periocular tissue

Table 4 presents the results after injection into the ocular tissue. In all tissues examined, the degree of penetration into the eye depended on the concentration of CA4P introduced into the tissue surrounding the eye. The highest concentrations of the drug in the eye were achieved within the first hour after administration. Therapeutically relevant concentrations of the drug (> 1 μmol) were delivered to the retina and choroid proper at all doses administered. A high concentration of the drug was also observed in the sclera. Relatively little drug was found in the vitreous or plasma.

Table 4
Biological distribution of CA4P after injection into the ocular tissue Eye tissue sample Dose (mg / ml) C _max (μmol) Vitreous body one hundred 3.35 10 0.34 one 0,03 Retina one hundred 169 10 54 one 4.4 Actually choroid one hundred 1,040 10 74.5 one 14.3 Sclera one hundred 3,366 10 280 one eighteen Plasma one hundred 12.3 10 0.79 one 0,07

(v) Local formulations

Topical gels and solutions have been developed for use as topical formulations suitable for topical delivery of CA4P to the surface of the eye. Local solutions (1, 3 and 10%) were directly prepared in 0.9% NaCl (Aguettant, Lyon, France) and sterilized with a 0.2 μm filter (pH from 6.4 to 8.5, osmolarity from 290 to 459 mosmol / kg H ₂ O. Local gels (1, 3 and 10%) with low viscosity were prepared in 0.5% carboxymethyl cellulose (Sigma Aldrich Chimie, St. Quentin Fallavier Cedex, France) with 0.9% NaCl. Physicochemical specifications of each gel are shown in table 5.

Table 5
Local gel compositions CA4P 1% gel CA4P 3% CA4P gel 10% CA4P gel Test Specification Result Specification Result Specification Result pH 7.4-8.1 7,796 7.7-8.4 8,203 8.1-8.8 8,491 Osmolarity 330-370 354 290-330 315 475-515 495 Viscosity (MPa · s) 30-80 49 30-80 58 50-100 71

Local formulations were applied to the surface of the right eyes at an applied dose of 5 μCi in a volume of 50 μl. Corneal samples were taken instead of sclera. Samples were taken after 0.5, 1, 6 and 24 hours.

Table 6 shows the results of a study of the biological distribution after the administration of each local CA4P gel composition. The highest concentrations of the drug in the eye were achieved within the first hour after administration. Therapeutically relevant drug concentrations (> 1 μmol) were delivered to the cornea, retina and choroid proper with all three gel formulations. Relatively little drug was found in blood plasma.

Table 6
Biological distribution of CA4P after topical gel application Eye tissue sample Dose (% CA4P) C _max (μmol) Cornea 10 292 3 118 one 82 Watery moisture 10 13 3 8.2 one 2,4 Actually choroid 10 22.5 3 10.7 one 2,8 Retina 10 5.8 3 5.8 one 1,0 Plasma 10 1,63 3 0.56 one 0.09

Table 7 shows the results of a study of the biological distribution after the administration of each local CA4P formulation in the form of a solution. In all tissues examined, the degree of penetration into the eye depended on the concentration of CA4P in the solution of the composition. The highest drug concentrations in the eye were achieved within the first hour after administration. Therapeutically relevant concentrations of the drug (> 1 μmol) were delivered to the cornea with all three compositions in the form of a solution, while a dose of 10 mg / ml led to the delivery of a significant amount of the drug to the retina and the choroid itself.

Table 7
The biological distribution of CA4P after topical application of the solution Eye tissue sample Dose (% CA4P) C _max (μmol) Cornea 10 104 3 34 one 10 Watery moisture 10 4.1 3 1.9 one 0.6 Actually choroid 10 15.6 3 1.9 one 0.87 Retina 10 4.2 3 0.39 one 0.27 Plasma 10 1.27 3 0.84 one 0,07

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

Example 3. The introduction of CA4P in the eye by iontophoresis

CA4P can be ionized at physiological pH and therefore amenable to iontophoretic delivery. The efficiency of iontophoretic delivery of CA4P through the sclera was evaluated using an ophthalmic rabbit ophthalmic applicator (IOMED Inc., Salt Lake City, UT) composed of a 180 μl silicone receiving shell, the lining of which is a current distribution component made of silver foil coated with silver chloride, connecting current-conducting wire and one layer of a hydrogel-impregnated matrix of polyvinyl acetal, on which CA4P (10 mg / ml) was applied. The contact surface area of the applicator was 0.54 cm ² . The applicator was placed on the sclera in the right eyes of New Zealand white rabbits (3-3.5 kg, n = 6 for each treatment) in the upper blind sac near the limb with the front edge 1-2 mm distal to the corneal scleral junction. DC anode iontophoresis was performed by each applicator at 2, 3, and 4 mA for 20 minutes using Phoresor II (PM 700 (IOMED Inc., Salt Lake City, UT). Passive iontophoresis (0 mA for 20) was used as a control minutes) After treatment, the animals were euthanized and eyes were enucleated 30 minutes after treatment, rinsed with tap water and frozen at -70 ° C. The retina and the choroid proper were separated from these samples.

Quantification of CA4P, CA, and diethylstilbestrol internal standard (Sigma Chemical Company) was performed using approximately 100 mg of tissue using a tandem mass spectrometry chromatography method (LC / MS / MS). An aliquot of the metal extract was injected into an SCIEX API 3000 3000 LC / MS / MS device equipped with an HPLC column. The peak area m / z 315-> 285 of the CA43 product ion and m / z 395-> 79 of the CA4P product ion were measured in comparison with the peak area of m / z 267-> 237 of the internal standard product ion. Quantitative analysis was performed using regression analyzes by the weighted (l / x) linear least squares method generated by the enriched calibration standards obtained immediately before each analysis session. The initial amounts of combretastatin were significantly higher than the range of quantification, and therefore they had to be extrapolated after analysis. As shown in table 8, the delivery of the total amount of combretastatin to the choroid itself and the retina is enhanced by iontophoresis by approximately 15 times when compared with passive delivery. These levels represent an excess of several thousand times over what is considered therapeutically relevant concentration for inhibiting tubulin binding (2-3 μmol). However, delivery to the retina / choroid proper was not current dependent.

Table 8
Iontophoretic enhancement of combretastatin delivery to the retina / choroid proper (mean ± SD) Treatment Amount of CA4P delivered (ng) Amount of CA4 delivered (ng) The total amount of delivered combretastatin (nmol / g) Gain 0 mA, 20 min <0.4 57 ± 37 1.6 ± 1.0 Not rated 2 mA, 20 min 1.4 ± 0.5 910 ± 630 27 ± 15 17 3 mA, 20 min 3.8 ± 1.5 710 ± 450 25 ± 17 16 4 mA, 20 min 7.2 ± 6.3 670 ± 440 24 ± 10 fifteen

Example 4. The treatment of neovascularization of the cornea by systemic administration of CA4P

To mimic pathogenic ocular angiogenesis, neovascularization of the eyes was induced by the administration of lipid hydroperoxide (LHP) by a corneal injection at a dose of 30 μg into the eyes of rabbits. After 7-14 days, ocular vessels formed in the injected eyes due to damage caused by LHP. Subjects were divided into two groups; animals from the first group were injected with combretastatin A4 disodium phosphate intravenously at a dose of 40 mg / kg once / day for 5 days, while a carrier without combretastatin A4 disodium phosphate was administered to the other group intravenously as a dosage of water for the same period of time. The eyes of animals of both groups were examined after 7 days. Vascular reduction of 40% or more was observed in the group treated with combretastatin A4 disodium phosphate, but it was not in another group.

Example 5. The treatment of neovascularization of the cornea through systemic administration of CA4P

To assess the ability of CA4P to inhibit corneal neovascularization, a rabbit corneal model was used in which neovascularization was induced by injection of linoleic acid hydroxide ("LHP") (Ueda et al., Angiogenesis, 1997, 1: 174-184). Injection of LHP into the corneal stroma stimulates localized production of angiogenic cytokines within the cornea. Blood vessels in the surrounding limb plexus respond to angiogenic stimulation by migration towards the LHP injection site. The therapeutic efficacy of systemically delivered CA4P was evaluated by measuring the length of these proliferating vessels.

Experimental methods:

As shown in Table 9 below, adult male New Zealand rabbits (2.7-3.0 kg) were injected with 10 μl of a suspension of LHP (60 μg) 5 mm from the upper limb in order to cause corneal angiogenesis. The vessels grew at a rate of 0.25 mm / day. Groups 2 and 4 were injected intraperitoneally ("ip") with CA4P (50 mg / kg) respectively after 3 and 10 days of vascular growth. Treatment groups 1 and 3 were injected with salt control on the 3rd and 10th days after LHP injection. Superficial photographs of the cornea were taken on days 0, 3, 6, 12, 17, and 28 after LHP injection. After each photographing session, corneal vessels were observed under an operational microscope, and a Castroviejo caliper was used to measure the length of the most prominent vessel.

In addition to measuring the longest vessel, histological analysis was performed on day 28 to evaluate the amount of extracellular matrix dissolved, the vessel wall thickness and the degree of branching of the vessels. Euthanized animals were enucleated and the vitreous was removed from each eye before fixation in 4% paraformaldehyde for 45 minutes and 0.2 M cacodylate buffer (pH 7.4) overnight. Eyes were placed in paraffin, sections were made with a thickness of 3 μm and stained with hemolysin and eosin.

Table 9
Experiment Log Group Sample size Treatment Day Scheme Euthanasia day one n = 4 Carrier (PBS) 3 4 times / day (5 days 28 2 n = 5 CA4P (50 mg / kg) 3 4 times / day (5 days 28 3 n = 2 Carrier (PBS) 10 4 times / day (5 days 28 2 days rest 4 times / day (5 days four n = 7 CA4P (50 mg / kg) 10 4 times / day (5 days 28 2 days rest 4 times / day (5 days

Results:

Tables 10 and 11 summarize the effects of CA4P on the length of blood vessels as a function of time-intervention and the number of treatment procedures. When treatment with CA4P was used for intervention within 3 days from the initial angiogenic stimulation (Table 10, group 2), the drug caused complete inhibition of new vessel growth. In contrast, vessels in the carrier control group continued to grow. The indicated effect can be qualified as suppression of angiogenesis or anti-angiogenic effect. When CA4P treatment was used for intervention 10 days after angiogenic stimulation (Table 11, Group 2), the effect was the same.

Table 10
Early intervention: Treatment begins on day 3 Group The length of the vessels (mm) on the 3rd day The length of the vessels (mm) on the 6th day The length of the vessels (mm) on the 12th day Media control
(group 1)
50 mg / kg
CA4P
(group 2)
P value 0.8 ± 0.12
0.6 ± 0.12 1.9 ± 0.04
0.7 ± 0.16
<0.001 3.7 ± 0.13
0.5 ± 0.61
<0.001

Table 11
Late intervention: Treatment begins on day 10 Group The length of the vessels (mm) on the 10th day The length of the vessels (mm) on the 12th day The length of the vessels (mm) on the 24th day Media control
(group 3)
50 mg / kg
CA4P
(group 4)
P value 4.0 ± 0.7
2.6 ± 0.29 5.4 ± 0.6
2.8 ± 0.49
> 0.05 6.4 ± 0.3
1.4 ± 0.46
> 0.05

Figv is a photograph of the surface of the eye treated with CA4P on the 28th day. This photograph further illustrates inhibition of vascular growth on day 28 after administration of CA4P compared to the carrier control eye of FIG. 3A.

The micrographs shown in FIGS. 4A and 4B (enlargement of H400) are examples of stained histological samples obtained from the same animals on day 28. In animals treated with the carrier (figa), the vessels were round and numerous. In contrast, in animals treated with CA4P (Fig. 4B), the vessels were narrow and less numerous. In addition, vascular regression data were observed at later stages of the intervention with CA4P administration (data not shown). It turned out that CA4P was able to reduce the width of the resulting vessels and significantly inhibit the germination of branches from the vessels, which indicates an additional effect aimed at the vessels.

Example 6. The treatment of neovascularization of the choroid proper on an experimental model of macular degeneration through systemic administration of CA4P

Neovascularization of the choroid proper is the main cause of severe vision loss in patients with age-related or wet macular degeneration. To study the ability of CA4P to inhibit vascular growth in the choroid itself, we tested a model of neovascularization of the choroid of the eye itself in mice. In this model, the researcher uses a krypton laser to create a wound on the C57BL / 6J mouse basal plate. Several burns were made in each eye. The burn caused a classic wound healing reaction that included neovascularization inside the choroid itself. This method of photocoagulation with a krypton laser has been described in Tobe et al., Am. J. of Pathology, 1998, 153 (5): 1641-6. In a subgroup of animals (n = 19), CA4P was systemically administered by ip injection at a dose of 100 mg / kg / day. Pathology and fluorescein angiography was used to identify the neovascularization surrounding the burn. Electron microscopy was used to measure the diameter of the lumen of the fenestrated network of new vessels inside the neovascular lesions of the choroid itself. Table 12 illustrates the effects of CA4P treatment on the mean vascular lumen area. In animals treated with CA4P, the vascular lumen area (mm ² ) was approximately 50% less when compared with animals treated with saline (n = 33). Statistical analysis of the results showed a high degree of significance.

Table 12
The average vascular lumen area in mice treated with CA4P and vehicle MOUSE CA4P (100 mg / kg) CA4P (20 mg / kg) CA4P (10 mg / kg) Carrier The number of mice in the group 19 eleven 10 33 The average area of the lumen of blood vessels (mm ² ) 0.0076115 0.0110057 0.011858 0.0129886 Standard deviation 0,0032669 0,0043229 0,0041457 0.0047336

Example 7. Treatment of neovascularization of the retina in a model of retinopathy of premature mice in mice through systemic administration of CA4P

The mammalian inner retina receives oxygen from the superficial retinal capillary bed. This capillary bed is located below the inner bounding membrane, which serves as the interface between the inner retina and the outer avascular vitreous. The pathology of neovascularization of the retina or retinopathy occurs due to ischemia-induced growth of a new vasculature outside the retinal internal bounding membrane and into the vitreous, causing severe loss of vision and often leading to retinal detachment. A well-characterized murine model of oxygen-induced retinal neovascularization closely mimics prematurity retinopathy ("ROP"), which manifests itself in prematurely born human infants and has characteristics common to various other retinopathies caused by ischemia, including diabetic retinopathy (Smith et al., Invest. Ophthalmol Vis. Sci. 1994, 35: 101-11). In this model, newborn mice are exposed to persistent hyperoxic conditions (75% oxygen for 7 days) that inhibit the development of the superficial retinal capillary bed. When a mouse is removed from the environment of pure oxygen and placed in relative oxygen hypoxia, the underdeveloped surface retinal capillary bed is unable to deliver sufficient oxygen to the retina. The retina responds to the lack of oxygen by producing angiogenic cytokines, which cause severe pathological effects. Localized production of angiogenic cytokines can cause germination of new vessels from the underdeveloped surface retinal capillary bed, which destroy the internal bounding membrane. The growth of aberrant blood vessels in the vitreous causes the formation of a significant amount of scar tissue and a retinal detachment caused by traction.

It is expected that treatment of a newborn CA4P mouse immediately after its removal from hyperoxic conditions may be an effective treatment for ROP. Retinal neovascularization can be quantitatively characterized by counting the number of chemically stained nuclei of the penetrating endothelial cells in the tissue section of the retina of animals treated and not treated, in accordance with existing methods (Majka et al., Invest. Ophthalmol. Vis. Sci. 2001, 42: 210 -fifteen). It is expected that the number of nuclei of cells penetrating the internal limiting membrane should be significantly reduced in the eyes of animals treated with CA4P.

Example 8. The treatment of eye tumors in a mouse model of retinoblastoma by subconjunctival administration of CA4P

A murine transgenic model was used, in which mice with the large T-antigen SV-40 develop a bilateral retinoblastoma resembling retinoblastoma in children. In this model, tumors first appear at the age of 4 weeks and develop in a stable and reproducible manner (Hayden et al., Arch Ophthalmol. 2002; 120 (3): 353-9). In one experiment, animals of 12 weeks of age (n = 48) were treated with a single subconjunctival injection of 20 μl of CA4P (100 mg / ml) in the right eye only. Control mice (n = 8) were treated with balanced saline ("BSS"). Eye samples were taken by enucleation on days 1, 3, 7, 14, 21 and 28 after treatment (n = 8 eyes per sample). Samples were fixed, placed in paraffin, serial sections were made and stained with hematoxylin, eosin after a preliminary PAS reaction (Schick reaction). Each tissue sample was examined to identify the histopathological reaction of the tumor vessels. As shown in FIG. 5A, a significant decrease in intratumoral vascularization was evident on days 1, 3, and 7.

In a separate experiment, the administration of animals (n = 48) was treated with 6 serial subconjunctival injections of CA4P 2 times / week at concentrations of 100, 10, 1, 0.1 or 0.01 mg / ml in a volume of 20 μl (n = 6 eyes per sample) . The control group (n = 6) received serial subconjunctival injections of BSS. Eye enucleation was performed 28 days after treatment and was examined to detect a decrease in tumor volume. FIG. 5B illustrates a dose-dependent effect of CA4P on the vascular volume of a tumor in comparison with a control. An intratumoral vasculature was absent at therapeutic dose levels above 10 mg / ml, and toxicity data were not observed at any time point or therapeutic dose.

Other options for implementation

It should be understood that, although the invention has been described in connection with its detailed description, the foregoing description is intended to illustrate and not to limit the range of claims of the invention, which is defined by the range of claims of the attached claims.

It should also be understood that the drawings are not necessarily scaled, they are merely conceptual.

Claims (29)

1. A method of treating eye neovascularization associated with a non-malignant vascular proliferative disease, comprising administering a pharmaceutically effective dosage of combretastatin A4 prodrug, which is about 0.1 mg / ml to about 100 mg / ml for non-systemic administration and about 1 mg for systemic administration / kg to about 100 mg / kg. 2. The method according to claim 1, in which these eye diseases may include neovascularization of the retina, neovascularization of the choroid itself, diabetic retinopathy, retinopathy of prematurity, neovascularization of the cornea and macular degeneration. 3. The method according to claim 1, where the specified pharmaceutically effective amount is administered non-systemically into the eye of the specified subject by injection into the vitreous humor, injections under the conjunctiva, injection into the periocular tissue, injection into the subepiscular space, through eye drops, through iontophoresis and through an eye implant and / or eye liner. 4. The method according to claim 1, wherein the pharmaceutically effective dosage is administered parenterally. 5. The method according to claim 1, wherein said subject is a mammal. 6. A pharmaceutical drug for the treatment of ocular diseases selected from non-malignant vascular proliferative disorders, comprising a therapeutically effective amount of a combretastatin A4 prodrug, which is about 0.1 mg / ml to about 100 mg / ml for non-systemic administration and about about 100 mg / ml for systemic administration 1 mg / kg to about 100 mg / kg, together with a pharmaceutically acceptable carrier, excipient, diluent or adjuvant for administration to a subject in need thereof. 7. The pharmaceutical drug of claim 6, wherein said eye diseases may include neovascularization of the retina, neovascularization of the choroid proper, diabetic retinopathy, premature neonatal retinopathy, corneal neovascularization and macular degeneration. 8. The pharmaceutical drug according to claim 6, where the specified pharmaceutically effective amount is administered non-systemically into the eye of the specified subject by injection into the vitreous humor, injection under the conjunctiva, injection into the ocular tissue, injection into the subepiscular space, through eye drops, by means of iontophoresis and through the eye implant and / or eye liner. 9. The pharmaceutical drug according to claim 6, wherein said pharmaceutically effective dosage is administered parenterally. 10. The pharmaceutical drug according to claim 6, wherein said subject is a mammal. 11. A pharmaceutical composition comprising a therapeutically effective amount of a tubulin-binding agent of the combretastatin A4 prodrug, which is about 0.1 mg / ml to about 100 mg / ml for non-systemic administration and about 1 mg / kg to about 100 mg for systemic administration / kg, for administration to a subject suffering from an ocular disease, wherein the ocular disease is a non-cancerous vascular proliferative disorder. 12. The pharmaceutical composition according to claim 11, where the specified pharmaceutically effective amount is administered non-systemically into the eye of the specified subject by injection into the vitreous humor, injection under the conjunctiva, injection into the ocular tissue, injection into the subepiscular space, through eye drops, through iontophoresis and through an eye implant and / or eye liner. 13. The pharmaceutical composition of claim 11, wherein said ocular diseases may include neovascularization of the retina, neovascularization of the choroid proper, diabetic retinopathy, premature retinopathy, neovascularization of the cornea and macular degeneration. 14. The pharmaceutical composition according to claim 11, wherein said therapeutically effective amount of combretastatin A4 prodrug is administered parenterally. 15. The pharmaceutical composition according to claim 11, wherein said subject is a mammal. 16. A pharmaceutical dosage form that includes a therapeutically effective amount of a combretastatin A4 prodrug, which is about 0.1 mg / ml to about 100 mg / ml for non-systemic administration and about 1 mg / kg to about 100 mg / kg for systemic administration and a pharmaceutically acceptable carrier or excipient for the treatment of ocular diseases constituting a non-cancerous vascular proliferative disorder. 17. The pharmaceutical dosage form according to clause 16, wherein said eye diseases may include neovascularization of the retina, neovascularization of the choroid proper, diabetic retinopathy, prematurity retinopathy, neovascularization of the cornea and macular degeneration. 18. The pharmaceutical dosage form according to clause 16, where the specified pharmaceutically effective amount is administered non-systemically into the eye of the specified subject by injection into the vitreous humor, injection under the conjunctiva, injection into the ocular tissue, injection into the subepiscular space, through eye drops, through iontophoresis and through the eye implant and / or eye liner. 19. The pharmaceutical dosage form according to clause 16, wherein said therapeutically effective amount of a combretastatin A4 prodrug is administered parenterally. 20. The pharmaceutical dosage form according to clause 16, wherein said subject is a mammal. 21. A method for treating ocular diseases, which are non-malignant vascular proliferative disorders, by administering a combretastatin A4 prodrug into the eye of a subject in need thereof in a dose sufficient to achieve a combrestatin A4 prodrug concentration in the eye in the range from about 1 nmol to about 100 mmol of intraocular fluid . 22. The method according to item 21, in which these eye diseases can include neovascularization of the retina, neovascularization of the choroid itself, diabetic retinopathy, retinopathy of prematurity, neovascularization of the cornea and macular degeneration. 23. The method according to item 21, in which the prodrug of combretastatin A4 is injected into the eye of the specified subject by injection into the vitreous body, injection under the conjunctiva, injection into the ocular tissue, injection into the subepiscular space, through eye drops, through an eye gel, through an eye ointment, by iontophoresis and by means of an ophthalmic implant and / or ocular insert. 24. A pharmaceutical composition for topical administration to an eye of a subject affected by an ocular disease, wherein the ocular disease is a non-cancerous vascular proliferative disorder comprising a prodrug of combretastatin A4 in an amount of about 0.1 mg / ml to about 100 mg / ml together with a pharmaceutically acceptable carrier , excipient, diluent or adjuvant for administration to a subject in need thereof. 25. The pharmaceutical composition according to paragraph 24, where these eye diseases may include neovascularization of the retina, neovascularization of the choroid itself, diabetic retinopathy, retinopathy of prematurity, neovascularization of the cornea and macular degeneration. 26. The pharmaceutical composition according to paragraph 24, wherein said composition is administered non-systemically into the eye of said subject. 27. A pharmaceutical composition for topical administration to an eye of a subject affected by an eye disease, the composition comprising in suspension, emulsion or solution:
(a) an amount of CA4P in the range of about 0.1 mg / ml to about 100 mg / ml;
(b) approximately 5 mg / ml carboxymethyl cellulose;
(c) approximately 9 mg / ml NaCl;
(d) has a final pH in the range of about 6.6 to 8.6;
(e) has an osmolarity in the range of from about 291 to 492 mosmol / kg H ₂ O and
(f) a viscosity in the range of about 50 to 66 MPa · s. 28. The pharmaceutical composition of claim 27, wherein said ocular diseases may include neovascularization of the retina, neovascularization of the choroid proper, diabetic retinopathy, premature neonatal retinopathy, corneal neovascularization and macular degeneration. 29. The pharmaceutical composition according to item 27, where the specified composition is administered unsystematically in the eye of the specified subject.

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