Compositions and articles for reducing the effects of inflammation

<u style="single">Technical field</u>The present invention generally relates to the field of drug therapy, and with respect to the use of photodynamic therapy (PDT) to reduce or prevent inflammation caused by injured tissue, which injured tissue is an intestinal injury (eg, surgery). It may be due to accidental injury (eg, skin laceration, injury to joints and tendons, and treatment of burn victims). In a preferred embodiment, the present invention relates to the use of "low dose" PDT for the treatment of eye tissue, where the inflammation is due to the manipulation of the eye tissue, especially for the patient to recover from the required procedure. This is the case when presenting complex factors. Common applications include inflammatory eye disease and various types of eye surgery or laser treatment, such as transplantation, and filtered eye surgery commonly used in the treatment of glaucoma. In a particularly preferred embodiment, the present invention relates to prolongation of survival of filtration blebs to improve the outcome of filtration surgery.<u style="single">Background technology</u><u style="single">Inflammation in general</u>The four major symptoms commonly associated with inflammation are: (1) redness, (2) swelling, (3) fever and (4) pain, and any fifth major symptomatology is the function of the affected area. Is a loss. Injury triggers a complex sequence of events, many of which occur simultaneously and are interrelated in various ways, but it is known that small blood vessels are involved in the induction of inflammation in an important way. There is. In fact, inflammation is one of the body's valuable defense mechanisms and is usually thought to have three phases: the degenerative phase, the vascular phase, and the healing phase. ). Klein, "Defense Reactions in Action", Immunology, The Science of Self-Nonself Discrimination, See Chapter 14, 577-84 (1982). This disclosure is incorporated herein by reference. Specifically, in the degenerative phase, the affected cells (mainly epithelial cells and fibroblasts) swell, their cytoplasm becomes vacuolated, and their nuclei expand and fragment. Some of the platelets in the damaged blood vessels break down and release serotonin and other mediators that act on the sympathetic nerve endings. The vascular phase is a change in blood vessels, the widespread migration and activity of so-called inflammatory cells (granulocytes-especially neutrophils, lymphocytes, monocytes, and macrophages), as well as degenerated cells and cellular. Characterized by the removal of debris). The capillary network and posterior capillary veins become bloody, congested, and angry in active hyperemia. The number of capillaries also increases, so the inflamed tissue has a reddish appearance (sometimes called "redness"). Increased blood flow also brings the temperature of the inflamed area closer to that of aortic blood, which is hotter than the surrounding normal tissue, giving a feeling of heat. Upon injury, the injured tissue releases a substance known to be associated with histamine called the H substance. It is a mixture of histamine and serotonin released by mast cells in destroyed tissue. The H substance causes active dilation of blood vessels, and the endothelial cells of the dilated blood vessels separate from each other, resulting in a large void between the endothelial cells. The endothelium that lines the blood vessels is gradually covered with white blood cells, causing some of the fluid to escape into the surrounding tissue. A protein-rich fluid that leaks from blood vessels into surrounding tissue causes the tissue to swell. The leaked fluid also contains a substance that neutralizes bacterial toxins, which aids in the destruction of inflammatory factors. White blood cells, especially neutrophils and monocytes, move along the vessel wall until suitable voids are found that can escape into the perivascular structure and tissue space. Leukocytes attack dead and dying cells and either digest them intracellularly by phagocytosis or extracellularly proteolytic enzymes, which are released from their lysosomes when they themselves die. To digest by. The stimulus for leukocyte ejection is thought to come from the injured tissue in the form of a chemotactic factor. Platelets are another cell type that is greatly affected by tissue damage. Immediately after injury, platelets adhere to the vessel wall alone or in clumps. At the same time, fibrin fibers begin to appear, forming a fine network that aids in cell capture. The resulting blood clot pulls together the edges of the collapsed tissue. Intracellular and extracellular digestion of necrotic tissue by neutrophils and monocytes produces fluid, which is combined with serous material that is extruded from blood vessels. When an abscess forms, the tooth cavity is lined with a febrile membrane. It prevents the dissemination and growth of pathogenic microorganisms in the blood in bacterially infected wounds. In the first two phases of the inflammatory process, the foreign body is destroyed (eg, if the foreign body is an organism) or the surrounding tissue loosens (eg, the foreign body is a splinter). In the case of). In the healing phase, inflammation begins to subside; individual vascular and vascular patterns become normal again; and wound repair begins. The three main events in the repair process are (1) formation of new connective tissue by proliferation of fibroblasts; (2) epithelial regeneration; And (3) the growth of new capillaries. Even before sedation of inflammation, fibroblasts begin to migrate from the surrounding normal tissue, where fibroblasts normally exist in a dormant state, into the injured area. Fibroblasts migrate along fibrin strands in amoebic movement and distribute themselves throughout the healing area. Once fixed in place in the injured tissue, fibroblasts begin to synthesize collagen and secrete this protein, which arranges itself as a fiber. The fiber orients itself in the direction of highest stress on its major axis. As the collagen bundle grows and becomes firm, the fibroblasts gradually degenerate and adhere closely to this bundle, and the injured area transforms into scar tissue. Upon the formation of scar tissue, intact epithelial cells at the edges of the wound begin to proliferate and move toward the center of the injured area as a single sheet. As the inflammation subsides, there is a need for a direct supply of blood, and new blood vessels begin to grow in the wound. When looking at inflammation molecularly, it is known that many active compounds interact with each other in a complex manner. Among the cells damaged by injury, mast cells release mediators that trigger vasodilation in the early phase, which involves separation of endothelial cells and exposure of collagen fibers in the subepithelial layer. The intercellular void fibers formed in the blood vessels capture platelets and trigger the release of mediators from these cells. In addition to platelets, exposed collagen fibers also interact with plasma proteins that exude through the pores of the dilated blood vessel wall, which contain factors that trigger the blood coagulation cascade. These proteins also initiate a kinin-bradykinin cascade, producing bradykinin, which becomes involved in vasodilation and enhances vascular permeability and chemotaxis. The fourth molecular system, the complement cascade, can be activated by several stimuli: Damaged blood vessels, proteolytic enzymes released by damaged cells, membrane components of any of the involved bacteria, and antigen-antibody complexes. Some of the activated complement components act as motivational factors, causing leukocytes to flow into the inflamed area. Others promote phagocytosis and are involved in cell lysis.<u style="single">Inflammation of the eye</u>Glaucoma is an eye injury, where high intraocular pressure impairs an individual's vision. In the normal eye, aqueous humor is produced by the epithelial cells of the ciliary body. The ciliary body is located along the inner circumference of the iris (towards the inside of the eyeball). The function of this aqueous humor involves nourishing the cells of the eye and keeping the inside of the eye at positive pressure, which keeps the visual parts necessary for image formation in the correct spatial arrangement. It is necessary to do so, and is similar to the support structure of the camera body in a photographic camera. Aqueous humor is normally removed from the eye by filtration through the trabecular meshwork. The trabecular meshwork is an annulus located along the circumference in the angle between the iris and the cornea in the anterior part of the eye. Aqueous humor is typically drained into Schlemm's canal through micropores in the trabecular meshwork, and then the connector. Through the channel), this directs the aqueous humor into the episcleral vein and out of the eye. In the pathology of glaucoma, the outflow of aqueous humor from the eye is reduced, resulting in a sharp increase in intraocular pressure, damage to internal ocular tissue, and ultimately complete loss of vision. The therapeutic objectives in glaucoma treatment are always the same. That is, reducing intraocular pressure by either reducing the production of aqueous humor or increasing the amount of aqueous humor that is drained or "filtered" out of the eye. There are many ways to achieve this goal, but drug therapy is always the first to be tried. If drug therapy does not successfully control elevated intraocular pressure, other, more invasive techniques are used. For example, a laser procedure or a surgical procedure (intervention). Laser treatment involves trabeculoplasty, where a laser is used to burn a hole in the trabeculoplasty. Surgical techniques include: (1) trabeculectomy, which uses a metal probe or a "trabeculotom" to connect Schlemm's canal to the anterior chamber of the eye. In between, create an opening of approximately one-third of the circumference of the normal drainage angle; (2) trabeculectomy, which involves incising the trabeculectomy; And (3) iridectomy, which means removing part of the iris. A sclerostomy involves making an incision in the sclera with either a laser or a surgical instrument. Trabecular meshwork, irisectomy and scleral incision all involve the formation of filtration "blebs". Filtration vesicles are small sac in which excess ocular fluid diverges, facilitating excretion from the eye. Glaucoma filtration surgery is usually recommended for patients with progressive glaucoma injury and for patients who are at significant risk of disease progression at current levels of intraocular pressure. In patients with severe injury, intraocular pressure (IOP) can be reduced to less than 20 mmHg, and if it can be maintained below this level, the long-term prognosis is improved. Therefore, filtration surgery is usually highly recommended for patients with progressive injury and intraocular pressure above 18-20 mmHg. This surgery usually falls into one of two categories: (1) full thickness procedure or (2) guarded fisula procedure. More basically, the protective fistula method typically involves the following trabecular meshwork steps: a. Retracting step of the eyelid; b. Corneal ring (translucent tissue, eye Penetrates through the opaque cornea (representing the transition between the opaque cornea) and into the anterior chamber (separated by the colored iris and the transparent cornea that covers the iris) from the outside of the anterior part of the eye. The process of forming the opening of the cornea; c. At the position below the iris, the outer layer of the condyle is peeled off, and the triangular flap (the base of the triangle is the corneal ring); d. The process of entering the anterior chamber from the base of the corneal valve; e. The process of excising a part of the underlying trabecular meshwork to form a fistula or connecting tube; f. The process of excising a small piece of iris through the fistula. G. The step of closing the corneal valve with a suture; h. The step of suturing and closing the cornea; and i. A physiologically acceptable fluid (eg, a basic salt solution (BSS)) is injected into the anterior chamber through an external opening that penetrates the corneal ring (which was made in step b) and into the corneal ring. The process of elevating the vesicles formed along it to ensure that the fistula is not blocked and that the vesicles are leak-free. The full-thickness method differs in that an opening is created directly without a scleral valve to connect the anterior chamber and the subconjunctival space through the corneal ring. After removing the outer layer of the cornea, sclerectomy (cutting the edge of the tissue from the sclera at the annulus of the cornea), thermal incision (a shallow groove in the sclera, the surface of the annulus of the cornea) (Cut parallel to), laser scleral incision, or perforation. Stewart, Filtering Surgery--Techniques and Operative Complications, Clinical Practice of Glaucoma, Chapter 10, 333-61 (1990), the disclosure of which is incorporated herein by reference. It will be used. After surgery, the condition of the filter vesicles should be carefully monitored regularly. Initially, the vesicles are usually sufficiently elevated and separated from the sclera (elevated off). In many eyes, the area of origin of the avascular condition in the conjunctiva is usually shown around the fistula site on the first day after surgery. Avascular regions are identified by focusing on localized loss of capillaries and venules. However, when the anterior chamber is examined, a small amount of redness or redness may be present, indicating inflammation. IOP is usually less than 5 mmHg 1 week after surgery, but can be in the range of 6-10 mmHg. After initial testing, patients are typically initiated with antibiotic-steroid combinations. Stewart, "Postoperative Complications of Filtering Surgery", Clinical Practice of Glaucoma, Chapter 11, 363-90 (1990), this disclosure is incorporated herein by reference. From 2 to 4 weeks after surgery, the conjunctiva and vesicles become less inflamed, and the anterior chamber becomes "calm" with the amount of flare subsided. Also, as a result of scarring, the vesicles are usually slightly smaller. In addition, the vesicles generally continue to exhibit avascular areas, which can increase in size. IOP usually rises above 10 mmHg from 2 to 4 weeks after surgery. Stewart, "Postoperative Complications of Filter Surgery", Clinical Practice of Glaucoma, Chapter 11, 363-90 (1990). After 4 weeks of non-complicated postoperative course, the conjunctiva is usually little or no inflammation. Well-functioning vesicles typically maintain an avascular area and can be separated from the sclera either minimally or sufficiently elevated. In addition, the IOP should ideally stabilize between 10 mmHg and 15 mmHg during this period. Topical postoperative steroids are slowly tapered according to the amount of inflammation in the filter bleb and anterior chamber. If the vesicles retain blood vessels and inflammation, steroids are usually maintained, and sometimes even increased, to accelerate the recovery of inflammation in any anterior segment, thereby limiting scar formation. Stewart, "Postoperative Complications of Filter Surgery", Clinical Practice of Glaucoma, Chapter 11, 363-90 (1990). Unfortunately, during the early postoperative period after filtration surgery, patients can suffer from a variety of different complications. One of these is bleb failure. In many patients, vesicle failure occurs between 1 and 6 months after surgery, and the vesicle eventually loses control of intraocular pressure. Clinically, poorly functioning filter blebs usually have a smaller range, less elevation, and at least partial angiogenesis, and the IOP rises again above the normal range. Stewart, "Postoperative Complications of Filter Surgery", Clincal Practice of Glaucoma, Chapter 11, 363-90 (1990). Successful filtration surgery depends on how long the cells remain functioning after surgery. Patients typically develop cystic insufficiency either by blocking at the site of the fistula or by scarring at the interface between the conjunctiva and sclera. If the fistula site is blocked, one of several laser treatment techniques or conventional surgical techniques can be used. Unfortunately, even if the fistula is opened in this way, the aqueous outflow can be limited by the previous scarring of the vesicles to the sclera. If these procedures fail and the patient's IOP is not controlled by maximal medical treatment, it may be necessary to perform other filtration procedures elsewhere. If the fistula remains open but the vesicles are small, elevated IOP is probably the result of scarring between the conjunctiva and sclera, which remains the most common cause of vesicle failure. Stewart, "Postoperative Complications of Filter Surgery", Clinical Practice of Glaucoma, Chapter 11, 363-90 (1990). The ocular tissue, especially the conjunctival procedure, in filtration surgery inevitably causes inflammation and ultimately scarring. In general, the more procedures there are, the shorter the lifespan of the vesicles. Fibroblasts appear to play a decisive role in this process, as filtration surgery in high-risk patients for glaucoma often fails as a result of postoperative scarring. Katz et al., "Mitomycin C versus 5-Fluorouracil in High-risk Glaucoma Filtration Surgery", Ophthalmology, 102: 9, 1263-68 (1995). One major cause of glaucoma filtration failure is the presence of fibroblasts in subconjunctival tissue (Berlin et al., The Role of Laser Sclerostomy in Glaucoma Surgery, Current Opinion in Ophthalmology, 6: 102-114 (1995), and if surgery fails, it is usually due to the presence of fibroblast proliferation and scarring at the site of filtration (Mora et al., "Sponge 5 intraoperatively." -Trabeculectomy with Intraoperative Sponge 5-Fluorouracil, Ophthalmology, 103: 963-70 (1996)). In so-called "high-risk" patients, when a high proportion of cystic insufficiency due to fibrosis is present, treatment in combination with surgery to prolong the survival of filtered follicles is sometimes useful. Many techniques have been devised to reduce inflammation and scarring, which prolongs the function of filter vesicles produced by filtration surgery. This technique includes, for example, a simple finger massage of the eye periodically over a period of about 4 weeks after surgery. Pharmacological techniques have also been attempted to limit scarring by inhibiting the inflammatory response and blocking the formation of collagen at specific steps in its synthetic pathway. Corticosteroids are often used topically, either as eye drops or as subconjunctival injections, to help prevent vesicle scarring by inhibiting the inflammatory response and fibroblast proliferation. Stewart, "Silment Surgery_Surgery and Surgical Complications", Clinical Practice of Glaucoma, Chapter 10, 333-61 (1990). Topical steroids are usually continued to minimize scarring until the inflammation in the anterior segment is resolved. Stewart, "Postoperative Complications of Filter Surgery", Clinical Practice of Glaucoma, Chapter 11, 363-90 (1990). A typical treatment program may indicate that postoperative topical use is performed with rapid tapering every 3 hours for as long as 20 days. Araujo et al., "A Ten-year Follow-up on a Prospective, Randomized Trial of Postoperative Corticosteroids after Traveculectomy", Ophthalmology, 102: 1753-59 (1995). 5-Fluorouracil ("5-FU") is a fluorinated pyrimidine analog with anti-metabolic activity (a competitive inhibitor of thymidylate synthase), which also has an anti-fibrotic effect by reducing fibroblast proliferation. This prevents scarring of the filter vesicles. Typically, 5-FU is used when the prognosis for surgery is poor. Two years later, 5-FU showed a success rate of filtration surgery between 60 and 70%. 5-FU is usually given in a series of subconjunctival injections. However, in addition to the inconvenience and discomfort of frequent and repetitive postoperative injections, many serious complications of subconjunctival 5-FU have been reported, including epithelial defects, subepithelial scars, and Includes corneal ulcer formation, leak of conjunctival wounds, cystic leakage, suprachoroidal hemorrhage, retinal detachment and endophthalmitis. Thus, although 5-FU can prolong cell life, the incidence of corneal epithelial defects, scarring, and angiogenesis is also high due to the general toxicity of the drug. Stewart, "Silment Surgery_Surgery and Surgical Complications", Clinical Practice of Glaucoma, Chapter 10, 333-61 (1990). Khaw et al. Also, "Five-minute Treatment with Fluorouracil, Floxuridine, and Mitomycin Have Long-term Effect on Human Tenon's Capsule. Fibroblasts) , Arch. Ophthalmol., 110: 1150-54 (1992); Kupin et al., Adjunctive Mitomycin C in Primary Trabeculectomy in Phakic Eyes , Am. J. of Ophtalmology, 119: 30-39 (1995); See also Katz et al., Mitomycin C vs. 5-fluorouracil in high-risk glaucoma filtration surgery, Ophthalmology, 102: 9, 1263-69 (1995). Kay et al. Also, "Delivery of Antifibroblast Agents as Adjuncts for Filter Surgery-Part II: Delivery of 5-Fluorouracil and Bleomycin into Collagen Implants: Delivery of Antifibroblast Agents as Adjuncts to Filtration Surgery-Part II: Delivery of 5-Fluorouracil and Bleomycin in a Collagen Implant: Pilot Study in the Rabbit) ", Ophthalmic Surg., 17: 796-801 (1986); And Khaw et al., "Effects of Inoperative 5-Fluorouracil or Mitomycin C on Glaucoma Filtration Surgery in the Rabbit", Opthalmology; 100: 367-72 (1993). ) Also see. Several authors report that corneal edema due to accidental intraocular exposure can be prevented by using low concentrations, eg, 0.5 mL of 10 mg / mL 5-FU, in conventional subconjunctival injections. are doing. Chalfin et al., "Corneal Endothelial Toxic Effect Secondary to Fluorouracil Needle Bleb Revision," Arch. Opthalmol., 113: 1093-94 (1993). Others use 5-FU more safely and effectively by intraoperative administration with a sponge soaked with 50 mg / mL compound and by keeping the sponge in contact with the vesicle site for a short period of time. We are paying attention to what can be done. Mora et al., "Intraoperative Trabeculectomy with Sponge 5-Fluorouracil," Ophthalmology, 103: 963-970 (1996)). However, even thereafter, postoperative injection of replacement is required in some cases, and that injection is also associated with an undesired high incidence of corneal epithelial damage. Floxyuridine, the deoxyribose sugar of fluorouracil, is about 100 times more potent than fluorouracil in long-term inhibition of ocular fibroblasts and can therefore be administered in a single dose. However, the differences that cause cell death rather than inhibition are relatively small. Therefore, the use of floxiuridine is susceptible to the risk of normal tissues being exposed to relatively high doses of potential cytotoxic substances. Khaw et al., "5-minute treatment with fluorouracil, floxiuridine, and mitomycin has long-term effects on human tenone-capsulated fibroblasts," Arch. Ophthalmo 1., 110: 1150-54 (1992). Similar effects are also known with mitomycin or mitomycin C (MMC). Since MMC is more potent than 5-FU, MMC can also be administered in a single intraoperative application, typically with a contact time of approximately 1 to 5 minutes, followed by heavy lavage. MMC is an alkylated antiproliferative and is isolated from the fermentation filtrate of certain species of Streptomyces. It is an anti-fibrotic, anti-neoplastic antibiotic that prevents scarring of filter follicles by inhibiting the proliferation of fibroblasts. This is usually effective in reducing postoperative subconjunctival fibrosis and therefore tends to prolong the residual time of filter vesicles and reduce IOP. However, MMC is also cell-destructive at high concentrations and causes unwanted eye hypotonia (IOP <5 or 6 mmHg) in as many as one-third of patients treated thereby. Other unwanted side effects include conjunctival wound leakage, choroidal detachment, and hypotonic macular degeneration (hypotony maculopahy), with a probability of late cyst leakage of approximately 25%. Khaw et al., "5-minute treatment with fluorouracil, floxiuridine, and mitomycin have long-term effects on human tenone-capsulated fibroblasts," Arch. Ophthalmol., 110: 1150-54 (1992); Zacharia et al., "Mitomycin C. Ocular Hypotony after Trabeculectomy with Mitomycin C, Am. J. of Ophthalmology, l16: 314-26 (1993); Kupin et al., "Early trabeculectomy in Phakic Eye." Attached Mitomycin C in Resection, Am. J. of Ophthalmology, 119: 30-39 (1995); Katz et al., Mitomycin C vs. 5-Fluorouracil in High-Risk Trabeculectomy, Ophthalmology, 102: 9, 1263-69 (1995); Shin et al. "Adjunctive Subconjunctival Mitomycin C in Glaucoma Triple Procedure", Ophthalmology, 102: 10,1550-58 (1995); Nouri-Mahdavi et al. , "Outcomes of Trabeculectomy for Primary Open-angle Glaucoma," Ophthalmology, 102: 12, 1760-69 (1995); and Mora et al. See Trabeculectomy with 5-Fluorouracil, Ophthamology, 103: 963-70 (1996). One of the group of researchers even reported an increased incidence of scleritis with severe pain and scleral redness after topical treatment with MMC during trabecular meshwork. Fouman, "Scleritis after Glaucoma Filtering Surgery with mitomyc in C", Ophthalmology, 102:10, 1569-71 (1995). Liang et al., "Comparison of Mitomycin C and 5-Fluorouracil on Filtration Surgery Success in Rabbit Eyes", J. Glaucoma, 1: 87-93 (1992). Other chemists have reported the use of laser scleral incisions in addition to either 5-FU or MMC. Berlin et al., "The Role of Laser Sclerostomy in Glaucoma Surgery," Current Opinion in Ophthalmology, 6 : ll, 102-l14 (1995), which is an MMC administered by many different routes (subconjunctival injection, subconjunctival gel foam, topical eye drops, or absorbent sponge) in addition to laser treatment. It suggests that its use can be even more effective. However, the usual complications of corneal toxicity, wound leakage, chronic hypotonicity, choroidal detachment, and hypotonic macular degeneration are also mentioned. Beta-aminopropionitrite (BAPN) and D-penicillamine have been used to inhibit collagen fiber cross-linking, which keeps collagen immature after filtration surgery. It can help to, and as a result, limit the scarring of the vesicles. Early reports of postoperative topical BAPN ointment found that it retained IOP below 22 mmHg in 74% of patients. However, animal studies with both BAPN and D-penicillamine showed limited efficacy. Stewart, Surgery_Surgery and Surgical Complications, Clinical Practice of Glaucoma, Chapter 10, 333-61 (1990). For a discussion of bleomycin, Khaw et al., "Intraoperative effects of 5-fluorouracil or mitomycin C on glaucoma filtration surgery in rabbits," Ophthalmology; 100: See 367-72 (1993). For a discussion of cytosine arabinoside-impregnated polymers, Lee et al., "Effects of Cytosine Arabinoside-impregnated Bioerodible Polymers on Glaucoma Filtration Surgery in Rabbits", See J. Glaucoma, 2: 96-100 (1993).<u style="single">Photodynamic therapy</u>Photodynamic therapy (PDT) is known as an acceptable cancer treatment, which has many purposes, such as the treatment of solid tumors (eg, US Pat. Nos. 4,932,934 and 5,283,255); blood-derived targets. (Eg leukemia cells), immunoreactive cells (co-pending application numbers 07 / 889,707; 08 / 309,509, 08 / 374,158 and 08 / 174,211), and unwanted microorganisms (e.g.) Impairment of US Pat. No. 5,360,734; Prevention of restenosis (US Pat. No. 5,422,362); Diagnosis and treatment of specific neovascular eye disorders (co-pending application numbers 08 / 209,473, ibid.) 08 / 390,591 and 08 / 613,420); Removal of atherosclerotic plaques (co-pending application number 08 / 663,890); and prevention of transplant rejection (co-pending application number 08 / 371,707) No.) can be used for. PDT is involved in the topical or systemic application of light-absorbing photosensitizers (usually porphyrin derivatives), which selectively accumulate in target tissues. Irradiation with visible light of activation wavelength produces reactive oxygen species in cells containing the photosensitizer, which promotes cell death. For example, in the treatment of tumors, the photosensitization process is thought to produce singlet oxygen, which is an active derivative of molecular oxygen, which can oxidatively react with many specific sites in cells and tissues. As a result, tumor cells suffer irreversible damage at the subcellular level, especially in the cell membrane and mitochondria. In vivo, tumor destruction is the result of a complex interaction of multiple factors that affect the connective tissue framework that physically supports the tumor's limbs and the vascular tissue that nourishes the tumor. Zhou, "Mechanisms of Tumor Necrosis Induced by Photodynamic Therapy", J. of Photochem. And Photobiol., B. Biology, 3, 299-318 (1989). It is clear that photosensitizers are preferentially taken up and accumulated in tumor tissue, and that necrosis of some tumor-supporting cells is selectively and directly caused by PDT. However, vascular injury followed by tumor cell anoxia also participates in the PDT-induced tumor necrosis process. Especially in the latter event, PDT-induced tumor necrosis is believed to be the result of an acute inflammatory response to physicochemical changes in the vessel wall. Due to the rapid decline in blood supply and the onset of inflammatory edema in the tumor, photoinjured neoplastic cells become hypoxic or even anoxic and eventually necrotize. This overall injury process is enhanced by the release of vasoactive or tissue-soluble substances (eg, histamine, proteases and acid phosphatases) from photodamaged mast cells and neutrophils in the tumor matrix. Is also associated with the inflammatory process. Zhou, "Mechanism of Tumor Necrosis Induced by Photodynamic Therapy", J. of Photochem. And Photobiol., B. Biology, 3, 299-318 (1989). It has been recognized that the acute inflammatory phase normally induced by PDT in acceptable cancer treatment protocols is a double-edged sword. Experimental tumor model studies have shown that after PDT is administered, protein and neutral lipid-rich exudates infiltrate the extracellular space and perinecrotic vital cells (hypoxic cells). Accumulates against the "wall" of the necrotic cells, indicating that it sticks to the "ghost" of necrotic cells. From the point of view of positive cancer treatment, the inflammatory exudate can help deliver the protein-bound photosensitizer to the inner region of the tumor. This is otherwise difficult to reach inward. On the other hand, this flow of inflammatory exudate can also carry oxygen and nutrients, thereby helping to nourish the cells involved in the wound repair process. Therefore, it is undeniably recognized that the development of PDT-related inflammatory conditions often complicates the treatment of cancerous tumors. Freitas, "Inflammation and Photodynamic Therapy", J. Photochem. And Photobiol., B: Biology, 8: 340-41 (1991). Several studies have been conducted to achieve anti-fibrotic effects in PDT related to glaucoma filtration surgery using tin ethyl etiopurpurin ([SnET2]) as a photosensitizer. Specifically, rabbits that received subconjunctival injection of SnET2 underwent filtration surgery and then postoperatively irradiated with light. Hill et al., "Photodynamic Therapy with Tin Ethyl Etiopurprin as an Alternative Anti-Fibrotic Treament Following Glaucoma Filtering Surgery", Photochem. Photobiol, 61 Suppl., 68S, TPM-E9 (1995); and Hill et al., "Photodynamic Therapy (PDT) for Antifibrosis in a Rabbit Model of Filtration" Surgery) Investigative Ophthalmology and Visual Science, 36: 4, S877 (1995). However, in this preliminary study, the authors did not provide any control data, thus determining how much the treatment of Hill et al. Actually extended the lifespan of the filtered follicles compared to the untreated follicles. That is difficult. In addition, Hill et al. Disclosure that more than 3 hours have passed since the photosensitizer was injected prior to the surgical and irradiation steps, which means that the photosensitizer is absorbed by the tissue associated with the injury. Gives enough time to be done, but also the photosensitizer spreads to other non-target areas of the eye. The authors report that a large transient avascular conjunctival area was generated and this avascular area was not confined to the filter vesicles until completely 4 weeks after surgery, so an undesired large area of the eye. Is clearly affected by this procedure. Given the well-known potential destructive necrotic effects of PDT in other applications, it is necessary to reduce or prevent inflammation to the extent and to the extent that pharmacological activity can be reliably controlled.<u style="single">Disclosure of invention</u>Surprisingly, with proper selection of non-toxic photosensitizers that are rapidly absorbed by the injured tissue but in the absence of light, PDT can have anti-inflammatory and beneficial anti-inflammatory effects. It has now become clear that this is even useful for delicate tissues such as the ocular region. This is a particularly surprising finding in view of past teachings that PDT actually causes an inflammatory response rather than having the ability to reduce or prevent inflammation. Specifically, it has now been discovered that the effects of inflammation originating from injured tissue can be reduced or prevented by low doses of PDT. Specifically, the methods of the invention for reducing or preventing such inflammation are: a. Contacting injured or pre-injured tissue with a photosensitizer capable of penetrating the tissue. The step of obtaining the desired degree of biodistribution in less than an hour; and b. Tissues thus contacted in tissue exposed to light having a wavelength absorbed by this photosensitizer. Includes the step of exposure for a time sufficient to reduce or prevent inflammation but not long enough to cause necrosis or erythema of the exposed tissue. The methods of the invention are particularly advantageous when the injured tissue is highly sensitive to further injury or inflammation (eg, ocular tissue). This is because the appropriate photosensitizer itself is not antiproliferative or cytotoxic to delicate tissues without active irradiation. Moreover, because most photosensitizers are non-toxic to human tissues unless activated by light, and because the photosensitizers of the present invention can penetrate into injured tissue relatively quickly. The degree of pharmacological activity can be easily controlled both by the degree of irradiation and by either the degree of physical contact with the photosensitizer or the concentration at the time of its irradiation (eg, in the bloodstream). .. As a result, the therapeutic effect of the present invention is more easily regulated than known pharmacological antifibrotic techniques. In another embodiment, the invention relates to a composition for reducing or preventing the effects of inflammation emanating from injured tissue. Photosensitizers from about 1 μg / mL to about 2 mg / mL, which can penetrate the injured or pre-injury tissue, resulting in the desired degree of biodistribution in less than about 1 hour, and; b. Includes pharmaceutically acceptable carriers. In yet another embodiment, the invention relates to an article for reducing or preventing the effects of inflammation emanating from an injured tissue: a. The article may penetrate the injured tissue, or pre-injury tissue, as a result. Includes a photosensitizer; and b. Adsorbent applicator, which gives the desired degree of biodistribution in less than an hour.<u style="single">[Simple explanation of drawings]</u>FIG. 1 is a graph showing the percentage of residual filter follicles of each rabbit in the four groups. FIG. 2 is a graph showing the differences between the groups regarding the range of vesicles on each examination day up to the 12th postoperative day. FIG. 3 is a graph showing the differences between the groups regarding the height of vesicles on each examination day up to the 12th postoperative day. FIG. 4 is a graph showing the difference in conjunctival edema on the filter bleb on each examination day up to the 12th postoperative day. FIG. 5 shows a typical green porphyrin formula useful in the methods, compositions, and articles of the present invention. FIG. 6 shows the structures of four BPD-type compounds that are particularly useful as the photosensitizer of the present invention.<u style="single">Detailed description of the invention</u>The term "inflammation" in this application refers to a series of changes that follow an injury in the body. Injury is caused by physical agents (eg, excessive heat or cold, pressure, ultraviolet or ionized irradiation, cutting or rubbing); by a wide range of inorganic or organic chemicals; or by biological agents (eg, viruses, bacteria). , And other parasites).<u style="single">Photosensitizer</u>A "photosensitizer" is a chemical compound that, when exposed to light of a wavelength that can be absorbed by a photosensitizer, absorbs light energy to produce the desired physiological effect (eg, a controlled anti-inflammatory effect). Is. Preferably, the photosensitizers of the present invention have an absorption spectrum that is in the wavelength range between 350 nm and 1200 nm, more preferably between 400 nm and 900 nm, and most preferably between 600 nm and 800 nm. It has, where the absorption spectrum can be adjusted to the desired penetration in a manner known per se. In general, another property of the photosensitizer, which is of particular importance in the practice of the present invention, is the relative lack of cytotoxicity due to the lack of photochemical effects and the targeting between specific cells and the photosensitizer. Rapid excretion from tissues due to the absence of specific interactions. The photosensitizers of the present invention can penetrate into the injured tissue to be treated in less than an hour and produce the desired degree of biodistribution, any suitable for photodynamic therapy (PDT). It can be a photosensitizer. Whether this criterion is met by potential photosensitizer candidates can be easily and quickly determined by the following simple tests: 1. Living cultured cells (preferably from suspended growth cultures; any; Cell lines are suitable). 2. The photosensitizer to be tested is added to the cells at a concentration of 1-3 ug / mL in the presence of 10% serum. 3. Centrifuge, then remove excess photosensitizer drug by the following various incubation periods (eg, 5, 15, 30, and 60 minutes). 4. The cells are washed with phosphate buffered saline and lysed by freezing and thawing. 5. Measure the concentration of the tested photosensitizer in the cell lysate by fluorescence against the appropriate standard. A group of particularly strong photosensitizers is Levy et al., U.S. Pat. No. 5,171, Includes green porphyrins described in detail in issue 749 (issued December 15, 1992, which is incorporated herein by reference). The term "green porphyrin" refers to a porphyrin derivative obtained by reacting a porphyrin nucleus with an alkyne in a Diels-Alder reaction to obtain monohydrobenzoporphyrin. Typically, green porphyrins promote the reaction in only one of the two available conjugated non-aromatic Diels structures present in the protoporphyrin IX-ring system (rings A and B). It is selected from the group of porphyrin derivatives obtained by the Diels-Alder reaction of acetylene derivatives with protoporphyrins. The structure of some typical green porphyrins is shown in Fig. 5. As shown in formulas 1 and 2 of FIG. 5, the Diels-Alder reaction first forms cyclohexadiene (referred to herein as "hydrobenzo") fused to the A or B pyrrole ring. Rearrangement of the π system of the hexadiene ring forms compounds of formulas 3 and 4, and reduction provides compounds of formulas 5 and 6. However, for practical reasons, the compounds of formulas 5 and 6 are preferably made by performing the previously discussed Diels-Alder reaction with corresponding olefins that are replaced with conventional acetylene compounds and are therefore more reduced. Form formation Generates a porphyrin ring structure. These compounds are represented in Formulas 1-6 with hydrogen occupying the nitrogen in the ring. However, it should be understood that metallized forms in which the cation is replaced with one or both of these hydrogens can also be used. The preparation of green porphyrin compounds useful in the present invention is described in detail in US Pat. No. 5,095,030. For convenience, the term hydromonobenzoporphyrin derivative abbreviation- "BPD"-is commonly used to represent the compounds of formulas 3 and 4 of FIG. Compounds of formulas 3 and 4 and mixtures thereof are particularly preferred. R as shown in Figure 5 Described in detail in No. 030. For convenience, the term hydromonobenzoporphyrin derivative abbreviation- "BPD"-is commonly used to represent the compounds of formulas 3 and 4 of FIG. Compounds of formulas 3 and 4 and mixtures thereof are particularly preferred. R as shown in Figure 5¹, R², R³, And R⁴Is a non-interfering substituent that does not noticeably affect the activity of the compound in the methods and compositions of the present invention. More specifically, the term "non-interfering substituent" means a substituent that does not diminish the ability of green porphyrins to act as photosensitizers that can be absorbed by injured tissue and exert pharmacological effects in less than an hour. Used as For the compounds in Figures 5 and 6, generally R¹And R²Are independently electron-withdrawing substituents or any other activating substituents that are sufficiently electron-withdrawing to increase the Diels-Alder reaction rate, which arise from both the A and B rings. It can, but preferably occurs in only one ring. Suitable R¹And R²Examples of groups are carvalcoxy (2-6C), alkyl (1-6C) sulfonyl or aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and -CONR.⁵CO- (here R⁵Includes aryl (6-10C) or alkyl (1-6C)). R¹And R²One of them may also be hydrogen, as long as the other is an electron-withdrawing substituent strong enough to promote the Diels-Alder reaction. Most commonly, R¹And R²Is a carbalcoxy group, preferably a methyl or ethyl carboxy ester. The preferred compound is R¹And R²Is the same, and is carvalcoxy, especially carboethoxy. As used herein, the term "carboxy" is -COOH, as defined by convention, while "carbalkoxy" stands for -COOR (where R is alkyl). "Carboxylalkyl" represents the substituent -R'-COOH (where R'is alkylene). "Calvarcoxylalkyl" stands for -R'-COOR, where R'is alkylene and R is alkyl or alkanol. In general, "alkyl" refers to the saturated hydrocarbyl moiety of a linear or branched chain of 1 to 6 carbon atoms (eg, methyl, n-hexyl, 2-methylpentyl, t-butyl, n-propyl, etc.). "Alkylene" is the same as "alkyl" except that the group is divalent rather than monovalent. "Aryl" represents an aromatic cyclic group such as phenyl, naphthyl, pyridyl and the like. The aryl groups of the invention are optionally substituted with 1-3 substituents, which are independently halos (eg fluoro, chloro, bromo, or iodo); lower alkyls (1-4C); and lower alkoxys (eg, fluoro, chloro, bromo, or iodo). It can be selected from the group consisting of 1 to 4C). The "aryl" or "alkylsulfonyl" group is of formula-SO₂Has R, where R is an alkyl or aryl as defined above. R³Are independently ω-carboxyalkyl groups (2-6C), or salts, amides, esters, or acylhydrazones thereof, or alkyl (1-6C). Preferably R³Is 2-carboxyethyl or an alkyl or alkanol ester thereof, and R⁴Is vinyl. However, most of these embodiments are preferred because of the availability of natural porphyrins rather than required by consideration of biological effects. R as shown in Figure 5¹-CCR²Formed by reaction with the protoporphyrin-IX ring system (where R³Is a protected form of 2-carboxyethyl, such as 2-carbomethoxyethyl or 2-carboethoxyethyl, R⁴Is-CH = CH₂The addition product is a compound of formulas 1 and 2. The compound of formula 1 results from the addition to the A ring, and the compound of formula 2 results from the addition to the B ring. A convenient starting material for the green porphyrin compounds of the present invention comprises naturally occurring porphyrins, wherein R.³Is-CH₂CH₂COOH, -CH₂CHRCONR₂, Or CH₂It is either CHRCOOR (where R is alkyl (1-6C) or alkanol (1-6C)). However, R³The very nature of is usually not related to the progress of the Diels-Alder reaction or the effectiveness of the product obtained, unless it contains a π-bond conjugate with the π-bond of the ring. Therefore, R³Can be any one of a wide variety of groups, such as lower alkyl (1-4C); and ω-carboxyalkyl (2-6C) and their esters and amides. R³Substituents can also be substituted with hydroxy groups; halogens (such as fluoro, chloro, bromo, or iodine); or other non-reactive substituents. R³Is-CH₂In the case of CHR-COOR, it has been found to be advantageous to hydrolyze or partially hydrolyze the esterified carboxy group. Typically, R³Hydrolysis at the -position is conveniently R¹Or R²It occurs much faster than hydrolysis at the ester group of. Moreover, the solubility and biodistribution properties of the resulting compounds are more desirable than those of the non-hydrolyzed form. Hydrolysis yields a diacid or monoacid product (or a salt thereof). In the compounds of formulas 1 and 2, R⁴Is usually at least initially -CH = CH₂However, this vinyl group is easily R by addition of ring B or A of formula 1 or 2 to the vinyl ring substituent, or oxidation thereof.⁴Derivatized to other embodiments. Therefore, R⁴Can be one of a wide variety of substituents consistent with those formed by a simple addition reaction. For example, the additive as an example could be of the HX type, where H is R.⁴Added to the carbon adjacent to the ring to provide the -position and has the following equation:<img file="JP4012575B2_D0001.tif" />Thus, in one embodiment, one of the added substituents is hydrogen, and the other is hydrogen; halo (such as fluoro, chloro, bromo, or iodine); hydroxy; lower alkoxy; amino; amide; Sulfhydryl; or selected from the group consisting of organosulfides. For example, the Markovnikov addition of water provides a substituent structure similar to the hematoporphyrin ring system for related rings. Vinyl groups are also R⁴-CH as a substituent at the -position₂It can be oxidized to obtain OH, -CHO, or -COOH, or salts or esters thereof. The addition or oxidation product itself can also be substituted if the added substituent is a functional leaving group. For example, if Br is a substituent, it is -OH, -OR (where R is alkyl (1-6C) as described above), halo-NH.₂, -NHR, -NR₂Can be replaced by parts such as. Therefore, in general, R⁴Is a vinyl group-CH = CH₂Represents any substituent that is easily converted by cleavage or addition, and additional substituents formed by the reaction of a good leaving group with the addition moiety. However, preferably R⁴Is: Vinyl (-CH = CH)₂);-CHOR^4', Here R^4'Is H or alkyl (1-6C) and -CH as needed₂Substituted with hydrophilic substituents such as OH; -CHO; COOH or -COOCH₃Like-COOR^4';-CH (OH) CH₃Or-CH (OCH)₃) CH₃Like-CH (OR^4') CH₃;-CH (OR)^4')-CH₂OR^4';-CH (OH) CH₂OH; -CH (SCH)₃) CH₃Like-CH (SR^4') CH₃And its diflufide;-CH (NR)^4') CH₃;-CH (CN) CH₃;-CH (Pyridineium bromide) CH₃;-CH (COOR)^4') CH₃;-CH (COOCR)^4') CH₃;-CHBrCH₃Like-CH₂(Halo) CH₃; Or-CH (halo) CH₂(Halo). Alternatively, R⁴Can be an organic group of less than 12 carbon atoms resulting from direct or indirect derivatization of vinyl. Or R⁴May provide an additional porphyrin or porphyrin-related ring system, such as a group containing 1-3 tetrapyrrole-type nuclei of formula-LP as defined below. R⁴Is-CH = CH₂, -CH (OH) CH₃, -CH (Halo) CH₃, Or a group containing 1-3 tetrapyrrole-type nuclei of formula-LP as defined below, these compounds are preferred. As used herein, the term "tetrapyrrole-type nucleus" refers to the following tetracyclic backbone:<img file="JP4012575B2_D0002.tif" />Or their salts, esters, amides, or acyl hydrazone, which are highly conjugated. This includes the porphyrin system, which is effectively a fully conjugated system; the chlorin system, which is effectively the dihydro form of porphyrin; and the reduced chlorin system, which is the tetrahydro form of the conjugated porphyrin system. When "porphyrin" is specified, a fully conjugated system is intended. Green porphyrin is effectively a porphyrin-based dihydro form. In one embodiment, the substituent R⁴Contains at least one additional tetrapyrrole-type nucleus. The product compound of the present invention is a dimer or oligomer, wherein at least one of the tetrapyrrole-type ring systems is a green porphyrin. R⁴The link between the green porphyrin moiety at the -position and the added tetrapyrrole ring system can be by an ether, amine, or vinyl bond. R⁴Porphyrin ring systems with two available substituent positions (in both the A and B rings) corresponding to can be further derivatized as described below. R⁴If is "-LP", -L- is selected from the following group:<img file="JP4012575B2_D0003.tif" />And P is a porphyrin structure or any second R⁴It is a second green porphyrin of formulas 1-6 shown in FIG. 5, except that the group is substituted by L above. (If -L- is of equation (e) or (f) shown above, the ring system to which the double bond is connected corresponds to the following in the ring to which the double bond is connected, as shown. It is also understood to have a resonant system:<img file="JP4012575B2_D0004.tif" />The hydro-monobenzoporphyrins produced directly from the Diels-Alder reaction described above can also be isomerized to the BPD compounds of formulas 3 and 4 of FIG. The depiction of compounds 3 and 4 in Figure 5 is R.²The relative positions of the outer ring methyl groups (ring A of formula 3 and ring B of formula 4) with respect to the substituents are not shown. Both isomers are available. The compounds of formulas 3 and 4 are particularly preferred in the methods and compositions of the present invention. In addition, the Diels-Alder product is selectively reduced by treatment with hydrogen in the presence of a catalyst (eg, palladium on carbon) and corresponds to the Diels-Alder product on rings A and B, respectively, in Equation 5 of FIG. And could give a saturated ring analog as shown as 6. However, as explained above, a more general practice is to carry out a Diels-Alder reaction initiated with an olefin starting material instead of the usual acetylene starting material to obtain a more reduced form of the resulting porphyrin ring system. That is. Remaining vinyl substituent (R)⁴) For derivatization by conversion, and for compounds of formulas 1 and 2, and R³The description described above for the variability of is similarly applied to the compounds of formulas 3, 4, 5, and 6. A preferred embodiment of the green porphyrin of the present invention is one in which the Diels-Alder product has been rearranged and partially hydrolyzed. Even more preferable is R³The carvalcoxy group at the -position is also a compound of formulas 3 and 4 (BPD) that has been hydrolyzed or partially hydrolyzed. -Compounds of the invention, including COOH, can be prepared either in the form of free acids or salts with organic or inorganic bases. FIG. 6 shows four particularly suitable compounds of the invention contained by formulas 3 and 4, which are aggregated as benzoporphyrin derivatives, namely BPD-DA, BPD-DB, BPD-MA, and BPD-Mb. Shown in. These are hydrolyzed or partially hydrolyzed forms of the rearranged products of formulas 3 and 4, where R³One or both of the protective carboxyl groups in the group have been hydrolyzed. R¹And R²Since the ester group in is hydrolyzed relatively slowly, the conversion to the type shown in FIG. 6 is easily accomplished. The most suitable of these green porphyrin compounds is BPD-MA. In Figure 6, R³Is-CH₂CH₂COOR^3'And here R^3'Varies with individual compounds. For details, see R in BPD-DA.¹And R²Is Calval Coxy, R^3'Is hydrogen, and derivatization is in ring A. BPD-DB is the corresponding compound derivatized in Ring B. BPD-MA represents the partially hydrolyzed form of BPD-DA, and BPD-MB represents the partially hydrolyzed form of BPD-DB. Therefore, in these latter compounds, R¹And R²Is Calval Coxy, one R^3'Is hydrogen, and the other R^3'Is alkyl (1-6C). The compounds of formulas BPD-MA and BPD-MB can be homologous, where only C-ring carbalcoxyethyl, or D-ring carbalcoxyethyl, is hydrolyzed, or C and D-ring substituent hydrolysis. It can be a mixture of things. In addition, any mixture of two or more BPD-MA, -MB, -DA, and -DB can be used in the methods and compositions of the present invention. It should be noted that many of the compounds in Figure 5 contain at least one chiral center and can therefore exist as optical isomers. The methods of the invention may use compounds having both chiral carbon configurations, where the compound is supplied as an isolate of a single stereoisomer or is a mixture of enantiomers and / or diastereomers. .. Separation of the diastereomeric mixture can be accomplished by any conventional means. Enantiomer mixtures can be separated by any conventional technique, for example by reacting them with an optically active preparation to separate the diastereomers. It should be further noted that the reaction product can be a mixture of unseparated A and B cycloadditions, such as a mixture of formulas 1 and 2, or 3 and 4, or 5 and 6. Either a separate form (eg, formula 3 alone or 4 alone) or a mixture of any ratio can be used in the methods and compositions of the present invention. Furthermore, the dimer form of green porphyrin and the dimer and multimer form of the green porphyrin / porphyrin combination can be used to further absorb light on a per mole basis. The dimer and oligomeric compounds of the present invention can be prepared using reactions similar to those for dimerization and oligomerization of porphyrins themselves. Green porphyrins or green porphyrin / porphyrin conjugates can be made directly, or porphyrins are coupled and subsequently converted to the corresponding green porphyrins by the Diels-Alder reaction of either or both of the terminal porphyrins.<u style="single">Pharmaceutical composition</u>Typically, the photosensitizers of the present invention use a photosensitizer, typically one or more physiologically acceptable carriers, i.e., at an appropriate pH and desired purity at ambient temperature. It is formulated into a pharmaceutical composition by mixing with a carrier that is non-toxic to the recipient at a given dosage and concentration. Suitable compositions include those suitable for systemic or topical administration, including infusion, transmucosal administration, or transdermal administration. Preferably, the composition of the invention comprises from about 1 μg / ml to about 2 mg / ml photosensitizer, primarily depending on the dosage form. For topical administration, preferably about 0.1 to about 2.0 mg / mL is used. For systemic administration (eg, intravenous injection), the concentration of photosensitizer will preferably vary from about 0.3 to about 0.5 mg / mL. Preferably, the photosensitizer is used alone with water or with other pharmaceutically acceptable excipients (eg, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton). Disclosed in Pennsylvania (Gennaro, ed., 1990), which is incorporated herein by reference), administered in liquid, gel, or gelatinous solid pharmaceutical compositions. .. In the case of liquids, the pharmaceutical composition containing the photosensitizer can be a suspension or an emulsion. In particular, liposomal or lipophilic formulations are often desirable. The photosensitizer of the present invention can be contained within liposomes, adhered to its surface, or both. Suitable methods for preparing liposomes are well known in the art. Including green porphyrin compounds in such preparations, for example, Allison et al., US Pat. No. 5,214,036 (issued May 25, 1993) and Desai et al., Co-pending Application No. 08 / 489,850 (1995). (Submitted June 13, 2014), which are incorporated herein by reference. If suspensions or emulsions are used, suitable excipients include water, saline, dextrose, glycerol and the like. These pharmaceutical compositions may also contain small amounts of non-toxic aids (eg, wetting or emulsifying agents, antioxidants, pH buffering agents, etc.). The pH of the formulation depends largely on the particular use and concentration of the photosensitizer, but is preferably in the range of about 3 to about 8. Preferably, the photosensitizer is to occur at a pH value close to physiological levels to prevent the photosensitizer from adhering to the container in which it is contained, and activation of the photosensitizer. Is maintained at a neutral pH (eg, about 6.5 to about 7.5) to ensure. Therefore, pH 6. A suitable embodiment is a photosensitizer formulation in electrolyte that contains the salt buffer equilibrated in 5, but does not contain fetal bovine serum (FBS). The reason FBS is omitted is that it contains antigenic components that can exacerbate the inflammatory response. If a photosensitizer is attached to a container in which the pharmaceutical composition containing it is stored, the appropriate non-antigenic component (eg, human serum albumin) will be applied to the injured tissue to be treated as needed. It can be added in an amount that does not conflict with the attached photosensitizer. The photosensitizer may be combined with one or more immunosuppressive agents to enhance the anti-inflammatory effect on the injured tissue. As used herein, the term "immunosuppressive agent" refers to a substance that behaves to suppress or mask the T-lymphocyte response. It includes substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include: 2-amino-6-aryl-5-substituted pyrimidine; azathiopurine or cyclophosphamide; bromocryptine; glutaaldehyde; anti-idiotype antibody against MHC antigen; cyclosporin A; One or more steroids, preferably corticosteroids and glucocorticosteroids (eg, prednison, methylprednisolone, and dexamethasone); anti-interferon γ antibody; anti-cancer necrosis factor-α antibody; anti-cancer necrosis factor-β antibody Anti-interleukin-2 antibody; anti-cytogenic receptor antibody (eg, anti-IL-2 receptor antibody); heterologous anti-lymphocyte globulin; pan T (pan-T) antibody, preferably OKT-3 monoclonal antibody; against CD4 Antibodies: Streptkinase; Streptdolnase; Or host-derived RNA or DNA. The immunosuppressant may be supplemented or used in combination with the same or lower dosage as the photosensitizer, and may be administered simultaneously or separately, systemically or topically. The effective amount of such other agents is largely a matter of therapeutic judgment, and the amount of photosensitizer present in the formulation, type of injury, type of immunosuppressant, site of delivery, method of administration, It depends on the dosing regimen, other factors described above, and other factors known to the practitioner. However, the amount of immunosuppressive agent suitable for use with the present invention is typically less than that usually suitable for the treatment of similar injured tissue. If an immunosuppressant is used, it can be administered by any suitable means, parenteral and intralesionally (ie, locally in the injured tissue) if local immunosuppressive treatment is desired. )including. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, and subconjunctival administration. If the pharmaceutical composition of the invention should be applied topically, for example if it should be applied to injured tissue, use a viscous solution, such as a gel, rather than a non-viscous solution. Would be preferable. The gel may be a gelling agent such as a polysaccharide, preferably a water-soluble polysaccharide (eg, hyaluronic acid, starch, cellulose derivatives (eg, methyl cellulose, hydroxyethyl cellulose, and carboxymethyl cellulose, etc.), for example, a solution of the desired photosensitizer. )) Can be prepared by mixing. When the polysaccharide is present in the gel formulation, the usual use is in the range of about 1-90% by weight of the gel, more preferably in the range of about 1-20% by weight. Examples of other suitable polysaccharides for this purpose and determination of the solubility of polysaccharides are provided in European Patent No. 267, Seen in issue 017 (issued May 11, 1988), the disclosure of which is incorporated herein by reference. Examples of suitable surfactants include poloxamer surfactants, which are block copolymers of ethylene oxide and propylene oxide, alone or mixed with lipids such as egg lecithin, a series of molecules. Represents. Another example of an emulsion commercially available from Green Cross is Fluosol-DA 20%, which contains a poloxamer surfactant, perfluorodecalin and perfluorotripropylamine emulsified with Pluronic F-68. Perfluoro compound emulsions and their effects in mammals are described in more detail in Bollands et al., J. Pharm. Pharmacol., 39: 10-21-24 (1987), the disclosure of which is herein. It is used as a reference. The pharmaceutical composition of the present invention is preferably sterilized. Sterility is easily achieved by sterile filtration through a 0.2 micron membrane. Once formulated and sterilized, the composition may become unstable to oxidative degeneration. However, for example, lyophilized formulations for reconstruction containing BPD are suitable for storage. More detailed in 10-21-24 (1987), the disclosure of which is incorporated herein by reference. The pharmaceutical composition of the present invention is preferably sterilized. Sterility is easily achieved by sterile filtration through a 0.2 micron membrane. Once formulated and sterilized, the composition may become unstable to oxidative degeneration. However, for example, lyophilized formulations for reconstruction containing BPD are suitable for storage. More detailed in 10-21-24 (1987), the disclosure of which is incorporated herein by reference. The pharmaceutical composition of the present invention is preferably sterilized. Sterility is easily achieved by sterile filtration through a 0.2 micron membrane. Once formulated and sterilized, the composition may become unstable to oxidative degeneration. However, for example, lyophilized formulations for reconstruction containing BPD are suitable for storage.<u style="single">The mode in which the tissue is brought into contact with the photosensitizer</u>The reduction or prevention of inflammation of the present invention can form a strong association between the photosensitizer and the target tissue, while minimizing the concentration of the photosensitizer and, as long as it is practicable, the wound of the target. It is accomplished in a relatively direct manner by contacting the wounded tissue (or tissue that should or should be inflamed) with a photosensitizer under conditions that may limit the extent of contact with the tissue. .. The photosensitizer can be administered topically or systemically if the cells to be protected from inflammation are contained within a living intact animal. The photosensitizer can be administered by injection as long as the particular mode of injection allows for rapid clearance of the photosensitizer from the body. For example, intravenous injection is suitable. Alternatively, the photosensitizer may be applied or sprayed, for example, onto the surface of the tissue to be treated, or a patch or implant, which can typically be removed at the end of the scheduled photosensitizer contact time. It can be applied topically or transenterically through. If the target tissue to be protected from inflammation is vulnerable eye tissue, topical topical administration is preferred due to the limited nature of eye contact achievable with topical administration, which is of greater safety. Bring limits. In a particularly preferred embodiment, the photosensitizer of the invention is applied with the article of the invention, which includes a photosensitizer and an absorbent applicator. Absorbent applicators include any absorbent material that is sterile or can be sterile, easily releases the photosensitizer upon contact with wounded tissue, and does not chemically react with the photosensitizer. Preferably, the absorbent material is also inexpensive and disposable. Examples of suitable absorbent applicators include drug impregnated sponges and soft woven fabrics that do not produce lint. Drug impregnated sponges such as Weck cells are preferred absorbent applicators. When such an applicator is used, it is preferably saturated with the pharmaceutical composition of the invention and during or immediately after the wound (eg, during surgery). , Applicable topically to the target tissue. The contact process should be carried out at a wide range of temperatures, avoiding only high temperatures sufficient to denature or otherwise adversely affect the wounded tissue, and low temperatures sufficient to minimize cellular uptake of the photosensitizer. Can be done over. Preferably, the contact step is carried out at a temperature in the range of about 5 ° C to about 40 ° C, preferably in the temperature range of about 15 ° C to about 37 ° C, and most preferably at an ambient temperature.<u style="single">dosage</u>In the methods of the invention, the subject is administered a certain amount of photosensitizer, or a mixture of photosensitizers, in a single or several doses. The photosensitizers of the present invention have the nature of inflammation to be prevented or alleviated, the species and pharmaceutical conditions of the subject, the presence of any other drug in the body of the subject, the purity and chemical form of the photosensitizer, It is administered in a manner consistent with good pharmaceutical practice, taking into account the mode of administration, the expected rate and degree of absorption, and other factors known to the practitioner. A therapeutically effective amount of the photosensitizer is an amount that is effective in significantly reducing the proliferation of fibroblasts upon irradiation with light and thus the inflammatory response and the undesired effects that may be associated with inflammation (eg, of vascular distribution). Increase and / or improve scar tissue formation). Photosensitizer dosing changes with the target tissue, either intravenously orWhen administered systemically, it is limited by animal weight and optimal blood levels. A suitable systemic dose per dose is typically less than about 1.0 mg / kg body weight, preferably about 0.25 to 0.75 mg / kg per dose, most preferably a single dose. It is about 0.15 to about 0.50 mg / kg per dose. Systemic dosing of BPD as a photosensitizer exceeds 0.3 mg / kg only under unusual circumstances. These dosage ranges are intended to be suggestive and should not be considered limiting. This is because the individual reactions of a particular subject also change. Optimal systemic blood level equivalents can be established, depending on the photosensitizer and the form of administration, but it is difficult to do so. This is because the photosensitizer is preferably discharged very quickly. Therefore, there can be a dramatic difference between the concentration of photosensitizer in the bloodstream at the moment of injection and the concentration at the time of treatment with light. For example, the BPD concentration at the moment of intravenous injection can be in the range of about 1-10 mg / mL, while at the time of light irradiation it can only be in the range of 0.5-0.05 ug / mL. By topical administration, typically no photosensitizer can be detected in the blood. When administered topically or systemically, dosing is best described by the concentration of the composition and the length of contact with the target tissue. Generally effective concentration ranges for photosensitizers are from about 0.1 to about 10 mg / mL, preferably from about 0.1 to about 5 mg / mL, and most preferably from about 0.25 to about 2. It is 0 mg / ml. This contact, as appropriate, comprises applying the composition to the surface of one or more wounded tissues together with the pharmaceutical composition of the present invention. In general, local contact with the photosensitizer takes at least 1 minute, preferably no more than 5 minutes, and even more preferably about 1-2 minutes. The contact time depends on factors such as the concentration of photosensitizer in the composition, the tissue to be treated, and the particular type of composition. After the planned contact time with the photosensitizer, preferably the excess photosensitizer is removed from the treatment area. When the photosensitizer is administered systemically, the photosensitizer is selected to have not only rapid pharmacokinetic properties, but also sensitivity to rapid clearance from the body. When the photosensitizer is administered topically, preferably the excess is a physiologically acceptable chemically inert fluid (eg, conventional saline or BSS (basic salt solution)). It is removed by irrigation or effluent with, or by washing with water or some other solvent. Again, these protocols are not intended to be limiting in terms of the wide range of changes allowed in the design of the protocol. Following the step of contacting the wounded or pre-wounded tissue with the composition comprising the photosensitizer of the present invention, the tissue is subjected to irradiation with light having a wavelength absorbed by the photosensitizer and is exposed to inflammation. Guide mitigation or prevention. The term "low dose PDT" in the present application refers to a dose that does not cause overt cell damage, necrosis, or erythema, but represents only an anti-inflammatory effect. Since the total PDT dose depends on the combination of the photosensitizer dose and the irradiation light dose, the low dose PDT is relatively in combination with the high photosensitizer dose and the low light dose, or on the one hand, relatively. It can be administered in combination with low photosensitizer doses and high light doses. The latter low photosensitizer / high light combination can also be achieved by administration of a relatively high dose of photosensitizer followed by an unusually long "incubation" time before irradiation with light. Therefore, a wide range of conditions that produce relatively low dose PDTs all as a whole are suitable for the present invention. Similarly, photosensitizer dose, contact time, And a wide variety of different combinations of dosage forms are suitable. However, the following general guidelines may be useful. Short contact (less than 1 hour) with a high photosensitizer dose (eg, topically applied 2 mg / mL) is generally equivalent to a low dose of photosensitizer (eg, 0.15 mg / kg administered intravenously). Is. However, delaying light irradiation to a later time (eg, longer than 3 hours) after photosensitizer administration, even after high doses of photosensitizer have been administered intravenously, also results in low dose PDT. obtain. This is because if the photosensitizer can be cleared quickly, only a very small amount can still be present in the tissue after 3 hours. Specific examples of "low dose PDT" include: Topical application or injection of less than 2 mg / mL benzoporphyrin derivative (BPD) photosensitizer, which takes less than 10 minutes with the target tissue. Stay in contact; Injective administration of BPD <0.15 mg / kg with irradiation after any time of BPD administration; or 0.15 to 0.50 mg / with irradiation after 6 hours of BPD administration Intravenous administration of kg of BPD; combined with irradiation under the following conditions: 15 J / cm applied for 0-3 hours after administration of the photosensitizer²Less than; or 100 J / cm applied later than 6 hours after administration of the photosensitizer²Until. Any light (eg, about 380 to about 850 nm, preferably about 400 to about 700 nm) that the photosensitizer absorbs (ie, suitable for use with wounded tissue) during the irradiation step is the photosensitizer and desired. Can be used depending on the depth of tissue permeation. For general anti-inflammatory applications, light in the visible part of the electromagnetic spectrum (eg, red light, blue light, or even UVA light) can be used. Light with wavelengths shorter than 400 nm is acceptable but unfavorable due to the potential damaging effect of UVA light. Light with wavelengths longer than 700 nm is also acceptable, but is not particularly preferred because it is difficult to see and therefore it is almost impossible to make visible control of the irradiation. Red light is preferred for eye application. This is because it removes any potentially detrimental effects from the blue and UVA spectral ranges on the sensitive retina of the eye. Filtration Examples of particularly suitable procedures used during surgery) are: 1. Saturate the drug-impregnated sponge with a 2 mg / mL aqueous dispersion of liposome BPD; 2. Treat the BPD saturated sponge Place in contact with tissue for 2 minutes; 3. Remove excess BPD by washing with a plethora of sterile physiological saline or equilibrium salt solution; and 4. BPD-treated tissue, approximately 7-12 J / cm²Irradiate with the light of. At this point, a single protocol appears to be undesirable in all cases. However, typical protocols include either a single procedure or an initial procedure that is followed by 1 to 4 additional procedures as needed. Topical treatment with topical photosensitizer administration can be repeated every 3 or 4 days. However, with systemic administration of photosensitizers, repeated treatments are generally left for about a week or longer to avoid any unwanted effects of excess photosensitizer accumulation. The following examples are intended to illustrate the invention, but are not intended to limit the invention.<u style="single">Example</u><u style="single">Example 1</u><u style="single">-</u>Light dosing Filter surgery was performed on one eye of 6 normal rabbits. Weck cell sponges were saturated with an aqueous solution of 2 mg / mL photosensitizer benzoporphyrin derivative monobasic acid ring A (BPD-MA, also known as "BPD-verteporfin"). During surgery, saturated Weck cells were used to locally apply BPD-MA to the sclera and conjunctiva in the surgical area for 2 minutes. After washing the excess drug with BSS, both the sclera and conjunctiva are from red light with a wavelength of about 690 nm (this is from a light emitting diode (LED) located about 1 cm from the tissue to be irradiated. Reached) exposed. Each of the 6 rabbits used in this experiment had different doses (specifically 0, 3, 6, 12, 18, and 24 J / cm over a period of 30 seconds to 4 minutes).²) Received the light. Treated rabbits were followed 11-12 days after surgery to measure filter vesicle height, vesicle vascular distribution (signs of inflammation), and decreased intraocular pressure (IOP). The data obtained on days 5 and 11 are shown in Tables 1A and 1B below, respectively.<img file="JP4012575B2_D0005.tif" /><img file="JP4012575B2_D0006.tif" />The results showed that residual filter vesicles were longest in eyes treated with light at moderate range doses (ie, relatively low doses of drug and light (low dose PDT)). The data showed that certain levels of PDT were required, but higher doses were generally less effective than lower doses. Short incubation time with BPD and 12 J / cm²The combination of low doses of light was not expected to cause significant damage to the cells being treated. Nevertheless, the treatment had a definite pharmacological effect. Vesicle remnants were associated with a lack of inflammation, as indicated by avascularity and pale-colored vesicles. On the other hand, when light doses that were too low or too high were used, the reduced amount of vesicle height and intraocular pressure was reduced. Cell failure was accompanied by inflammation. Corresponding filter vesicles were compared. In control (untreated) vesicles, vascular distribution in the vesicles was prominent 3 days after surgery. Minimal vascular distribution and high ridges were observed in PDT-treated vesicles 4 days after surgery.<u style="single">Example 2-Time of PDT administration</u>The photosensitizers used in the examples were prepared as follows: Benzoporphyrin derivatives formulated on liposomes, monobasic acid ring A, BPD-MA or BPD-berteporfin, QLT Photo Therapeutics, Inc. Was supplied as a lyophilized powder and reconstituted with sterile distilled water immediately before use. A 3 mm section of Weck cells was saturated using BPD reconstituted at 1.98 mg / mL. In the control group, Weck cells were saturated with a basic salt solution (BSS). On day 0, full-thickness filtration surgery was performed on 48 randomly selected eyes in 48 rabbits (12 in each of the 4 groups). In each rabbit, the other untreated eye was served as a control. The filtration procedure was performed in the following manner: Each animal was anesthetized with a mixture of ketamine and xylazine. The eyelids were separated using a wire microscope. A fornix-based conjunctival valve was created in either the upper nose or the upper temporal quadrant. Following the creation of a fornix-based valve, BPD-MA (or BSS placebo) saturated Weck cells were placed in the sclera behind the margin on which the fistula should be created. The conjunctiva was placed over the Weck cells. Thus, Weck cells are located between the conjunctiva and sclera and are in contact with the superior sclera and tenon sheath for 2 minutes. Weck cells were then removed and the area was irrigated with BSS. Instruments and gloves were also rinsed before entering the eye and placed in the anterior chamber using a 1.0 mm trephine. The conjunctival valve was then secured to the margin using two 7-0 sutures. Immediately after surgery, each rabbit was irradiated with light having a wavelength of 690 nm for 2 minutes using the light source (Quantum Devices, Inc.) used in Example 1 located 1 cm from the eye. Light source, maximum output (100W / cm²), Which provides a full dose of approximately 7.2J. One drop of tobramycin was then placed in each eye after surgery. Tobramycin and prednisolone acetate were instilled in both eyes four times daily for one week after surgery. The control eye received the same photosensitizer and subconjunctivally irradiation as the operated eye, but did not form a fistula. Control eyes were used to test toxicity and as a criterion for detecting IOP reduction for operated eyes. The time of BPD-MA administration was varied as follows: Group 1: Surgery, placebo treatment; 48 hours post-surgery, placebo treatment; Group 2: Surgery, BPD treatment; 48 hours post-surgery, placebo treatment Group 3: Surgery, placebo treatment; 48 hours post-surgery, BPD treatment; Group 4: Surgery, BPD treatment; and 48 hours post-surgery, BPD treatment; BPD treatment 48 hours post-surgery on filtration vesicles Application of 3 mm sections of Weck cells saturated with 2 mg / mL aqueous BPD-MA solution (or placebo) placed on the conjunct for 2 minutes, followed by excess photosensitizer wash, and red LED light with a wavelength of 688 nm. Consists of 1 minute of irradiation. Following surgery, every 0 and 2 days after surgery, rabbits are examined under a slit lamp biomicroscope for filter bleb size, filter bleb height, conjunctival erythema across the vesicles, anterior chamber cells. The flare and anterior chamber depth were evaluated. Intraocular pressure measurement for IOP measurement following local anesthesia. Subjective eye discomfort assessments were also performed by measuring animal comfort and dietary habits with the grading scales shown below: 0: Normal behavior 1: Shake, tilt, strabismus 2: Scratch the eyes 3: Residual lesion-making / self-cutting (on the nail) vesicles were assessed by vesicle size and height, and by IOP as a comparison to the control eye. The degree of inflammatory response was measured by erythema across the filter bleb and recorded on a scale of 0-3. Rabbits were sacrificed when cystic insufficiency was recorded (ie, when the intraocular pressure in the operated eye was equal to that in the control eye, and the filtration bleb flattened). The graph in Figure 1 shows the percentage of rabbit filter bleb remaining in each of the four groups evaluated in this study. The mean residual time (± SD) was as follows; 1 group: 10.3 ± 8 days; 2 group: 23.8 ± 12 days; 3 group: 10.1 ± 9 days; and 4 group: 23.2 ± 8 days. Statistical differences were observed between most groups (P <0.001), but not between groups 2 and 4 (P <0.05). Intraoperative BPD and light treatment (Groups 2 and 4) resulted in prolonged vesicle retention compared to placebo control (Group 1) or treatment only 48 hours after surgery (Group 3). Second treatment with BPD and light at 48 hours (Group 4) did not appear to have any additional effect. The Kruskal-Wallis test for non-parametric analysis was used to assess differences in filtration bleb retention time between groups. IOP was evaluated by the ANOVA trial and observed a tendency for BPD to be lower when given intraoperatively (P = 0.057). However, due to the problems associated with the tonometer used to measure IOP in rabbits, the most reliable parameters are vesicle size (Figure 2) and height (Figure 3), both of which are surgical. The efficacy of low-dose PDT treatment in medium was demonstrated. Measurements of erythema across the vesicles (shown in FIG. 4) showed a higher inflammatory response in groups 1 and 3, in which the vesicles disappeared early after surgery. Statistical differences were observed between the groups in Figures 2, 3 and 4 (P, 0. 001). Blister height and size, as well as other slit lamp characteristics, were analyzed by chi-square test. The opposite event was also analyzed by the χ-square test. The Cox Proportional Hazard Model was used to evaluate the parameters that most accurately predicted residual filtered blisters on each test day. When compared to previous rabbit studies investigating adjuvants for trabecular meshwork, the results show that BPD is compared to conventional controls, Ara-A and 5-fluorouracil, as shown below in Table 2. It was shown that the residual filter bleb was prolonged.<img file="JP4012575B2_D0007.tif" /><img file="JP4012575B2_D0008.tif" />The results also showed that BPD extended the lifespan of filtered blisters in rabbits longer than in some rabbits that received mitomycin C. The average residual time of studies with mitomycin C is longer, and this is because PDT does not clinically cause as much over-filtration as mitomycin C, but similar applications with greater safety. Consistent with the hypothesis that it has ease. This data clearly showed that longer blister residues were not associated with only erythema or minimal erythema, and thus with a reduced inflammatory response. The data also suggested that treatment with BPD and light at relatively low doses impeded the progression of inflammation, especially when administered intraoperatively. The opposite event was rare and was not particularly relevant to the use of photosensitizers. Within the first 4 days, fibrin clots were observed in 6 rabbits, 3 of which were in group 2 and 3 were in group 3. In each case, fibrin was dissolved without sequelae by the end of the first week. One rabbit died on day 0, which appeared to be a complication of anesthesia. No other adverse events were reported. On day 7, one rabbit from each group and two rabbits from each group after cyst failure were sacrificed for histological and transmission electron microscopy (TEM) analysis. Histological evaluation was performed after first fixing the cadaveric eye with 10% buffered neutral formalin. Eyes were treated, sectioned, and then stained with hematoxylin and eosin, as well as Masson trichrome. Specimens were tested in a masked fashion by a separate observer. Transmission electron microscopy was buffered with 0.1 M cacodylic acid containing 7% sucrose. This was done by immobilizing the tissue sample in 5% glutaraldehyde. Tissues were post-fixed with 2% osmium tetroxide after 1 hour and dehydrated through alcohol with a concentration gradient to 100% ethanol. A 2 × 5 mm tissue piece was then infiltrated with a catalytic epoxy resin. Thick part (0. 5 μm) was cut and stained with toluidine blue and examined under a light microscope to determine the appropriate region. A thin section (80 nm) was then cut, picked up on a copper grid and stained with uranyl acetate and lead citrate for TEM evaluation. These parts were inspected using a Hitachi H7000 transmission electron microscope. Light microscopy on day 7 post-surgery on filtration blebs that received BPD instead of placebo in surgery showed that fibroblasts and mild lymphocyte responses showed placebo on both days 0 and 2. It was shown to be present in the received rabbit. In addition, on day 7, these eyes showed some vascular proliferation and new collagen deposits. In contrast, rabbits (Group 3) who underwent BPD only on day 2 showed increased lymphatic channels in addition to fibroblasts, but no vascular proliferation. Filter follicles were noted to have a mild lymphocyte response in both eyes undergoing BPD by surgery. However, no increase in fibroblasts, vasculature, or lymphocyte channels was noted. At the end of this study (3 weeks post-surgery), eye fistulas undergoing BPD had very few lymphocytes and no vascular growth in the fistula. Thinned epithelium was observed in groups 2 and 4 both on day 7 and at sacrifice. However, this appears to be due to the vesicles raised by tear filling and associated disruption, as opposed to the toxic effects resulting from PDT treatment. In the control eye, no difference was observed between the placebo and the BPD-treated eye in the anterior segment ocular study, and the method or composition of the invention was clinically deficient in toxicity. Was shown (P> 0. 05). In addition, histological and transmission electron microscopy showed no evidence of toxicity or inflammation separate from the filtration site in either the surgical or control eye. Therefore, BPD-derived toxicity was not observed clinically, histologically, or by transmission electron microscopy.