Inhibition of angiogenesis

Certain simple chemical agents, referred to herein as nitrone related therapeutics or "NRTs", when administered to a patient susceptible to neovascularization (angiogenesis), can intervene and inhibit the disease's progress. Methods for therapeutically and prophylactically inhibiting angiogenesis by administering one or more NRTs are disclosed as are pharmaceutical compositions for use in such methods of treating. NRTs useful in these compositions and therapeutic methods are also disclosed.

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The invention relates to compounds, compositions and methods for inhibiting
 (preventing and treating) neovascularization (angiogenesis).
 2. Prior Work
 Malignant neovascularization (angiogenesis), particularly ocular
 neovascularization associated with macular degeneration, diabetic
 retinopathy and retinopathy of prematurity, as well as psoriasis,
 rheumatoid arthritis and solid tumors, is a serious medical condition.
 The most common cause of blindness in Americans over age 55 is age-related
 macular degeneration (AMD); for those under 40, diabetes is the most
 common cause of blindness. Neovascularization is the root cause of
 blindness in both cases. Neovascularization is the result of a compromise
 of the vascular bed supplying the retina, and may be regarded as a
 response to tissue ischemia (or hypoxia). Clinicians have long recognized
 the high probability of neovascularization in individuals who have lost
 part of the capillary bed due to diabetes, or who have experienced
 occlusion of a branch vein of the retina.
 The primary current treatment for neovascularization is destructive.
 Photocoagulation is used to reduce the volume of hypoxic tissue in
 diabetic retinopathy or to destroy vessels in AMD. Cryotherapy may be used
 to destroy hypoxic retina in infants. There is an urgent need for
 therapeutic intervention in these disease processes. No known therapeutic
 treatment can prevent neovascularization following loss of capillaries in
 diabetes, reduce the risk of further neovascularization in wet AMD, or
 offer reassurance to patients at risk because of heredity, diabetes, or
 age.
 Any progress toward therapeutic management and prevention of
 neovascularization will greatly reduce the social and economic impact of
 diabetes and AMD.
 One of the limitations of the newer therapeutic approaches to
 neovascularization that are under development, particularly those
 involving growth factors, is that they may also inhibit wound repair or
 the development of collateral vessels in mild occlusion of coronary
 arteries.
 STATEMENT OF THE INVENTION
 It has now been found that certain simple chemical agents, referred to
 herein as nitrone-related therapeutics or "NRTs", when administered to a
 patient susceptible to angiogenesis, can intervene and inhibit its
 progress.
 In one aspect this invention provides a method for inhibiting angiogenesis
 in a patient susceptible thereto by administering to that patient an
 effective angiogenesis-inhibiting dose of one or more NRTs.
 In a second aspect, this invention provides pharmaceutical compositions for
 use in such methods of treating. These compositions include one or more
 NRTs in a pharmaceutically acceptable carrier.
 In a third aspect, this invention provides NRTs useful in these
 compositions and therapeutic methods.
 DETAILED DESCRIPTION OF THE INVENTION

DESCRIPTION OF PREFERRED EMBODIMENTS
 This Description of Preferred Embodiments is broken into the following
 segments:
 The NRTs
 Pharmaceutical Preparations, Modes of Administration and Dosages
 Methods of Preparation of NRTs
 Description of Experiments
 The NRTs
 The NRTs which are employed in the practice of this invention are generally
 classed as spin-trapping agents. They include aromatic nitrones, including
 the best known nitrone, alpha-phenyl-N-t butyl nitrone ("PBN") and
 derivatives thereof; pyrolline N-oxides such as 5,5-dimethyl pyrroline
 N-oxide ("DMPO") and derivatives thereof; pyridyl N-oxide nitrones such as
 alpha-(4-pyridyl-1-oxide)-N-butyl nitrone ("POBN") and derivatives
 thereof. Examples of useful materials are described in U.S. Pat. No.
 5,622,994 and published PCT application number WO 92/22290, both of which
 are incorporated herein by reference.
 Among the NRT materials, aromatic nitrones are preferred. Aromatic nitrones
 are generally depicted by the formula
EQU X--C(R).dbd.N(O)--Y
 wherein X is an aromatic group, particularly a phenyl group or a phenyl
 group with at least one and particularly up to about three substituents
 selected from the following:
 lower alkyls of from one to about four carbon atoms, which may be linear or
 branched, and particularly methyls;
 lower alkenyls; halogens; haloalkyls; hydroxys; primary, secondary and
 tertiary amines; NOs; amides;
 lower alkoxyls, of from one to about four carbon atoms and particularly
 methoxyls;
 carboxylic acid functionalities, present as free acid --COOH groups or as
 suitable salts or esters such as lower alkyl esters of from one to about
 four carbon atoms and particularly methyl esters;
 sulfur-containing acid functionalities such as sulfates, sulfites and
 sulfonates, with the sulfates and sulfites being present as free acids or
 as salts.
 In this formula R is most typically hydrogen but can also be a lower alkyl,
 lower alkoxyl or the like, wherein "lower" has the one to four carbon atom
 meaning set forth above.
 In this formula Y is most commonly a one to twelve carbon alkyl group which
 may be straight chain or branched chain and which may be unsubstituted
 hydrocarbyl or may contain one or more heteroatoms substituents such as
 oxygen, sulfur, nitrogen or the like. These heteroatoms can be present as
 substituents in the Y group's main structural chain, for example as ether
 oxygens. Alternatively, the heteroatoms can be in the form of groups
 depending from the Y group main chain. Most commonly Y is from about two
 to about eight carbon atoms in size with no or one hydroxy or alkoxy
 substituents. Representative Y groups include methyl; ethyl; the propyls
 including n- and i-propyl; the butyls, especially t-butyl,
 heteroatom-substituted (such as hydroxy-substituted-) t-butyl and n-butyl;
 pentyls such as 1,1-dimethyl propyl and n-pentyl; the hexyls, heptyls and
 octyls.
 Some of these compounds include sulfate, sulfone, sulfoxide, sulfonamide or
 carboxylate groups. The sulfate groups can be present in an at least
 partially protonated acid form as a solid and in solution at low pH
 conditions. The weaker acid groups, such as carboxylates, are present as
 acids at somewhat higher pH's. These ionizable acid groups can also exist
 at higher pHs in an ionized salt form in combination with pharmaceutically
 acceptable cations. Most commonly, these cations are a monovalent material
 such as sodium, potassium or ammonium, but can also be a multivalent
 cation in combination with a pharmaceutically acceptable monovalent anion,
 for example calcium with a chloride, bromide, iodide, hydroxyl, nitrate,
 sulfonate, acetate, tartrate, oxalate, succinate, palmoate or the like
 anion; magnesium with such anions; zinc with such anions or the like. When
 reference is made herein to these sulfate or carboxylate groups or the
 like it will be understood to include the acid form as well as these salt
 forms, unless otherwise expressly stated. Often the salt forms are more
 stable than the corresponding free acids.
 Among these acid groups, the simple sodium, potassium and ammonium salts
 are most preferred with the calcium and magnesium salts also being
 preferred but somewhat less so.
 In the case of the other general types of NRTs, such as those based upon
 POBN or DMPO, the same types of substitutions can be employed as described
 with reference to the PBN type nitrones.
 Thus, in summary, the NRTs preferably used in this invention can be
 selected form the groups of aromatic nitrones of the formula
 X--C(R).dbd.N(O)--Y, wherein X, R and Y are defined above;
 PBN derivatives of the formula X--C(R).dbd.N(O)--Y, wherein X is a phenyl
 or a phenyl with substituents, and R and Y are defined above;
 DMPO and derivatives thereof of the general formula
 ##STR1##
 wherein A and B are each methyls or are each of the substituents listed
 with reference to the general aromatic nitrone formula; and POBN and
 derivatives thereof of the general formula
 ##STR2##
 wherein Y is as defined above, n is 0 to 4 and R.sup.2 is any of the
 substituents listed with reference to the aromatic nitrones.
 A group of most preferred NRTs is depicted in FIG. 1. These materials
 include the following compounds which are at times described using the
 noted compound references:
 (Compound Number 1) 3,5-dimethyl,4-hydroxyphenyl-N-n hexyl nitrone;
 (Compound Number 2) 3,5-dimethoxy,4-hydroxyphenyl-N-t butyl nitrone;
 (Compound Number 3) 2,4-disulfophenyl-N-ethyl nitrone, disodium salt;
 (Compound Number 4) 2,4-disulfophenyl-N-isopropyl nitrone, disodium salt;
 (Compound Number 5) 2,4-dihydroxyphenyl-N-t butyl nitrone;
 (Compound Number 6) 2,4-disulfophenyl-N-n butyl nitrone, disodium salt;
 (Compound Number 7) 2,4-disulfophenyl-N-1,1-dimethyl,2-hydroxyethyl
 nitrone, di-sodium salt; and
 (Compound Number 8) 2,4-disulfophenyl-N-t amyl nitrone, disodium salt.
 Of these, 3,5-dimethyl,4-hydroxyphenyl-N-n hexyl nitrone is the most
 preferred at this time.
 Pharmaceutical Preparations, Modes of Administration and Dosages
 Pharmaceutical preparations based upon the NRTs include one or more NRT in
 combination with a pharmaceutically acceptable carrier. The particular
 carrier employed will depend upon the mode of administration. Our studies
 provide evidence that the NRTs are effective in the treatment of
 angiogenesis when administered systemically, such as parenterally or
 orally. We also have evidence that the NRTs are active against
 angiogenesis when administered locally such as by intravitreal injection
 to the eye or topically to the eye via ointments, via eye drops of
 solutions or suspensions of particles or of liposomes or from a
 drug-releasing ocular insert.
 The compositions for oral administration can take the form of bulk liquid
 solutions or suspensions or bulk powders. More commonly, however, the
 compositions are presented in a unit dosage form to facilitate accurate
 dosing. Typical unit dosage forms include prefilled, premeasured ampules
 or syringes of the liquid compositions or pills, tablets, capsules or the
 like in the case of solid compositions. In such compositions, the NRT is
 usually a minor component (0.1 to say 50% by weight or preferably from
 about 1 to about 40% by weight) with the remainder being various vehicles
 or carriers and processing aids helpful for forming the desired dosing
 form. A liquid form may include a suitable aqueous or nonaqueous vehicle
 with buffers, suspending and dispensing agents, colorants, flavors and the
 like.
 A solid form may include, for example, any of the following ingredients, or
 compounds of a similar nature: a binder such as microcrystalline
 cellulose, gum tragacanth 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; a glidant such as
 colloidal silicon dioxide; a sweetening agent such as sucrose or
 saccharin; or a flavoring agent such as peppermint, methyl salicylate, or
 orange flavoring.
 In the case of injectable compositions, they are commonly based upon
 injectable sterile saline or phosphate-buffered saline or other injectable
 carriers (both aqueous and nonaqueous) known in the art. Again the active
 NRT is typically a minor component, often being from about 0.05 to 10% by
 weight with the remainder being the injectable carrier and the like.
 These components for orally administrable or injectable compositions are
 merely representative. Other materials as well as processing techniques
 and the like are set forth in Part 8 of Remington's Pharmaceutical
 Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which
 is incorporated by reference.
 When treating ocular neovascularization conditions, one can also administer
 the compounds of the invention topically to the eye in the form of an
 ocularly acceptable eye drop or suspension of particles or liposomes, from
 ointments or from a suitable sustained release form. Eye drops include a
 liquid carrier which is typically isotonic and sterile and also includes a
 suitable preservative and thixotropic material. Representative topical
 ocular preparations are described in chapter 2 "Pharmacokinetics: Routes
 of Administration", pages 18-43, of Ocular Pharmacology, Fifth Edition,
 William Havener, The C. V. Mosby Company, St. Louis, 1983, which is also
 incorporated herein by reference. As pointed out there, eye drop
 formulations may be based on simple aqueous vehicles or may employ more
 viscous vehicles such as thickened aqueous vehicles or nonaqueous
 materials such as vegetable oils or the like all with various buffers and
 salts to adjust the pH and tonicity to non-irritating levels. In these eye
 drop formulations the NRT can be present as a solute or as a suspension or
 in the form of liposomes based on phospholipids and the like.
 Ocular ointments include a gel or ointment base as described in Havener's
 Ocular Pharmacology. In these topical compositions the amount of NRT will
 range from about 0.05 to 10% by weight with the remainder being the
 carrier and the like. Typical concentrations for eye drops are 0.25-2% by
 weight.
 When direct delivery of NRT to the eye is desired, it may also be
 accomplished using sustained release forms or sustained release drug
 delivery systems. A description of representative sustained release
 materials such as soft contact lenses, soluble drug inserts and
 membrane-controlled diffusional systems, can be found in the incorporated
 materials in Havener's Ocular Pharmacology.
 The conditions treated with the NRT-containing pharmaceutical compositions
 may be classed generally as malignant neovascularization (angiogenesis)
 conditions. These occur with particular severity as ocular
 neovascularization associated with macular degeneration, diabetic
 retinopathy and retinopathy of prematurity. Angiogenesis is also observed
 in psoriasis, rheumatoid arthritis, and solid tumors. Each of these
 conditions is characterized by a progressive loss of function, such as
 vision, range of motion or skin integrity. The NRT compounds, when
 administered orally or by injection such as intravenously, can slow and
 delay and possibly even to some extent reverse the loss of function.
 Injection dose levels such as by intravenous administration for treating
 these conditions range from about 0.01 mg/kg/hr to about 10 mg/kg/hour.
 Such intravenous therapy might last for from less than a hour to as long
 as eight hours or more. A preloading bolus of from about 0.1 mg/kg to
 about 10 mg/kg or more may also be administered to achieve adequate steady
 state levels. The maximum total dose is not expected to exceed about 2
 g/day for a 40 to 80 kg adult patient.
 Many of the conditions treated are chronic in nature. With these chronic
 conditions, the regimen for treatment usually stretches over many months
 or years so oral dosing is preferred for patient convenience and
 tolerance. With oral dosing, one to five and especially two to four and
 typically three oral doses per day are representative regimens. Using
 these dosing patterns, each dose commonly provides from about 1 to about
 20 mg/kg of NRT, with preferred doses each providing from about 1 to about
 10 mg/kg and especially about 1 to about 5 mg/kg.
 In the case of treating angiogenesis associated with solid tumors, one can
 of course use systemic administration as just described. One can also use
 more localized delivery to the tumor site. This can be accomplished by
 close intra-arterial delivery where the artery chosen is one delivering
 blood to the tumor site where the angiogenesis is occurring. In the case
 of close intra-arterial administration one typically administers doses of
 up to about 10 mls, e.g. from about 0.25 to about 10 mls, containing from
 about 0.1 to about 10 and preferably from about 0.5 to about 5 mg/ml of
 active NRT. In the case of treating angiogenesis associated with solid
 tumors, the doses of NRT can be delivered daily or more often during the
 therapy period. One could also administer the active NRT by a continuous
 pumping into the arterial delivery route or continuously from a depot or
 other site within or near the tumor.
 Of course, one can administer an NRT as the sole active agent or one can
 administer it in combination with other agents, including other active
 NRTs.
 Methods of Preparation of NRTs
 Many of the NRTs employed herein are known compounds which may be purchased
 or which may be prepared by methods described in the literature. In
 addition, in the case of the NRTs which are simple nitrones, such as the
 PBN analogues described above as most preferred materials, these materials
 can be produced using a two step synthesis.
 In the first step, a commercially available nitroalkane (wherein the alkane
 corresponds to the R group present on the nitrogen in the final nitrone
 functionality) (for example 2-nitropropane or 2-nitrobutane) is converted
 to the corresponding hydroxylamine using a suitable reagent such as
 activated zinc/acetic acid, activated zinc/ammonium chloride or an
 aluminum/mercury amalgam. This reaction can be carried out in 0.5 to 12
 hours and especially about 2 to 6 hours or so at a temperature of about 0
 to 100.degree. C. in a liquid reaction medium such as alcohol/water
 mixture in the case of the zinc reagents or an ether/water mixture in the
 case of the aluminum amalgam reactant.
 In the second step, the freshly formed hydroxylamine is reacted in slight
 molar excess with a formyl-substituted aromatic compound which corresponds
 to the aromatic portion of the desired NRT. If the aromatic portion
 carries an acid substituent such as sulfonic acid or carboxylic acid
 functionality, this group will be present in the salt form. The position
 of the formyl group corresponds to the position of the nitrone in the
 final product, for example 2,4-dihydroxy benzaldehyde. The number (0, 1, 2
 or 3) and position (2, 3, 4, 5, or 6) of the substituents on the aromatic
 ring corresponds to the number and position in the final product. This
 reaction can be carried out at similar temperature conditions described
 with reference to the first step. This reaction is generally complete in 1
 to 48 hours and especially 10 to 24 hours.
 If the product so formed contains a sulfate, carboxylate or the like, such
 group is typically present as the salt. These salts can be converted to
 the free acid form by suitable acidification. Other salts can be easily
 formed by admixing the free acid in aqueous medium with the appropriate
 base, for example, KOH for the potassium salt, and the like.
 Description of Examples
 This invention will be further described with reference being made to the
 following experiments. These are intended to exemplify preferred aspects
 of this invention and are not to be construed as limiting its scope.
 Two in vitro experiments were conducted as Example 1 and 2 to determine
 whether or not NRTs showed promise as active agents against
 neovascularization.
 EXAMPLE 1
 In the first test, selected NRTs were tested for their ability to prevent
 lipid peroxidation of bovine retinal homogenates. Lipid peroxidation was
 induced by the addition of 2.5 mM Fe.sup.+2. NRTs were added to give
 concentrations of 10 and 100 .mu.g/ml which is approximately 40-400 .mu.M
 depending on the molecular weight of the NRT tested. Lipid peroxidation
 was measured by a TBARs assay. This assay is based on a modification of a
 fluorescent method reported by Yagi (Biochem. Med. 25:373-378(1981)). Of
 the eight NRTs tested, all were active as shown in FIG. 2 and in Table 1.
 EXAMPLE 2
 In the second test, selected NRTs were tested to determine their effect on
 preventing lipid peroxidation of isolated bovine retinal pigment
 epithelium cells. Lipid peroxidation was induced by the addition of 2.5 mM
 Fe.sup.+2. NRTs were added to give concentrations of 10 and 100 .mu.g/ml
 which is approximately 40-400 .mu.M depending on the molecular weight of
 the NRT tested. Lipid peroxidation was measured by a TBARs assay. This
 assay is based on a modification of a fluorescent method reported by Yagi
 (Biochem. Med. 25:373-378(1981)). Of the eight NRTs tested, five were
 active as shown in FIG. 3 and in Table 1.
 TABLE 1
 Activity of NRTs in In Vitro Assays
 Testing Ability to Inhibit Lipid Peroxidation
 Isolated
 Bovine Retinal Homogenate Bovine Pigment Epithelium
 Com- Con- Com- Con-
 pound centration Inhibition pound centration Inhibition
 1 10 .mu.g/ml 49% 1 10 .mu.g/ml 82%
 100 .mu.g/ml 100% 100 .mu.g/ml 31%
 2 10 .mu.g/ml 33% 2 10 .mu.g/ml 31%
 100 .mu.g/ml 67% 100 .mu.g/ml 69%
 6 10 .mu.g/ml 11% 5 10 .mu.g/ml 19%
 100 .mu.g/ml 53% 100 .mu.g/ml 44%
 7 10 .mu.g/ml 18% 6 10 .mu.g/ml 5%
 100 .mu.g/ml 35% 100 .mu.g/ml 30%
 5 10 .mu.g/ml 19% 3 10 .mu.g/ml 0%
 100 .mu.g/ml 28% 100 .mu.g/ml 18%
 4 10 .mu.g/ml 10% 7 10 .mu.g/ml 0%
 100 .mu.g/ml 19% 100 .mu.g/ml 0%
 8 10 .mu.g/ml 10% 8 10 .mu.g/ml 0%
 100 .mu.g/ml 17% 100 .mu.g/ml 0%
 3 10 .mu.g/ml 0% 4 10 .mu.g/ml 0%
 100 .mu.g/ml 27% 100 .mu.g/ml 0%
 EXAMPLE 3
 In one animal model for neovascularization, New Zealand white rabbits were
 treated with lipid hydroperoxide ("LHP"). In comparison to animals not so
 treated or treated with non-hydroperoxidized lipid (18:1 linoleic acid),
 these animals develop high degrees of neovascularization in their corneas
 and retina. A test material's effectiveness is measured by its ability to
 intervene in the neovascularization event.
 In one study, neovascularization of the cornea was examined. Vessels in the
 superior quadrant which were stimulated by LHP served as the positive
 control. They grew progressively to a mean length of 2.4 mm. There were
 approximately 20 separate vessels arising from the parent limbal vessels.
 Multiple branches were observed in this quadrant especially at the distal
 ends. Vessels in the center were always longer than at the edges. This was
 because neovascularization is a function of distance between the stimulus
 and the limbus. Thus, vessels were never observed in the inferior or
 intermediate quadrants. The controls using nonperoxidized linoleic acid
 (18:1) were essentially negative for vessel growth.
 To quantitate the neovascular response, Kodachrome slides taken from each
 group of animals were projected onto a screen and the entire vascular bed
 traced with an Opsiometer. This provided a cumulative index of total
 vessel proliferation at the various time intervals. These results are
 shown in FIG. 4.
 This study showed that Compound 1 was the most effective inhibitor of
 corneal neovascularization (38% at 3 days, 61% at 7 days, 67% at 10 days
 and 75-85% at 14 days post-exposure. Compound 6 was also effective; 42% at
 4 days, 37% at 7 days 46% at 11 days and 58% at 14 days post-exposure.
 Compound 2 showed anti-neovascular properties, but was the least effective
 of the drugs tested (17% at 4 days, 27% at 7 days, 33% at 11 days and 37%
 at 14 days post-exposure).
 From analysis of the slope and development of growth curves, it was
 determined that from 3 days until the end of the experiment, vessel
 proliferation was stopped completely by Compound 1. At 14 days, there was
 evidence for vessel retraction (from 7.5 mm to 6 mm). In contrast, vessels
 from Compound 6- and Compound 2-treated animals continued to grow in
 length and numbers until 10-11 days post-exposure. Compound 1 and Compound
 6 showed the greatest amount of vessel retraction (20 and 23%,
 respectively).
 Similar findings were obtained when the retina was examined. New vessels
 grew extensively in the LHP-treated animals without drug intervention.
 Numerous small branches were observed proximally and some were markedly
 dilated. At the distal end of vessels, there was extensive dilation and
 hemorrhage. In contrast, vessels in control animals injected with 18:1
 showed no edema, neovascularization, or hemorrhage.
 An animal treated with Compound 1 showed a reduction in neovascularization,
 but only slight effects on dilation and hemorrhage. Vasodilation edema and
 hemorrhage are prominent in an animal treated with Compound 2, however, no
 neovascularization was evident. Only dilation is observed in the retina
 treated with Compound 6.
 As shown in Table 2, Compounds 1, 2 and 6 all greatly retarded the
 neovascularization process. The degree of retardation ranged from 87.5%
 for Compound 2, to 75% for Compound 6, to 62.5% for Compound 1.
 TABLE 2
 Retinal Data
 Rabbit
 # CD or CS Treatment Dilation Hemorrhage
 Neovascularization RD Edema
 993 CD LHP - 14d + + +
 + +
 CS LHP - 14d + + +
 - -
 994 CD LHP - 14d - - -
 + +
 CS LHP - 14d + + +
 + +
 75% 75% 75%
 75% 75%
 996 CD 18: 1 - 14d - - -
 - -
 CS 18: 1 - 14d - - -
 - -
 997 CD 18: 1 - 14d - - -
 - -
 CS 18: 1 - 14d - - -
 - -
 977 CD 1 - 7d + + -
 + +
 CS 1 - 7d + + +
 + -
 978 CD 1 - 7d + - -
 - +
 CS 1 - 7d + - -
 - -
 979 CD 1 - 14d - - -
 + -
 CS 1 - 14d - - -
 - -
 980 CD 1 - 14d + + +
 - +
 CS 1 - 14d + + +
 - -
 75% 50% 37.5%
 37.5% 37.5%
 983 CD 2 - 7d - + -
 + -
 CS 2 - 7d + + -
 + -
 984 CD 2 - 7d + + +
 - +
 CS 2 - 7d + - -
 - +
 985 CD 2 - 14d + + -
 - +
 CS 2 - 14d + + -
 - -
 986 CD 2 - 14d - - -
 - -
 CS 2 - 14d - + -
 - +
 62.5% 75% 12.5%
 25% 50%
 989 CD 6 - 7d + + -
 - +
 CS 6 - 7d + - +
 - -
 992 CD 6 - 7d + + -
 + -
 CS* 6 - 7d + - +
 - -
 990 CD 6 - 14d - - -
 - -
 CS 6 - 14d - - -
 - -
 991 CD 6 - 14d + - -
 - -
 CS 6 - 14d + + -
 + -
 75% 37.5% 25%
 25% 12.5%
 *keratitis
 Compounds 1, 2 and 6 showed differing effects on neovascular-associated
 phenomena in the retina. Vasodilation, hemorrhage, retinal detachment and
 edema were observed in 75% of the eyes injected with LHP. In contrast, the
 control vehicle (18:1 linoleic acid) evoked none of these responses in
 either right (OD) or left eyes (OS). Compounds 1, 2 and 6 were ineffective
 in controlling dilation or hemorrhage, although Compound 2 reduced the
 incidence to 37.5%. Retinal detachment was also reduced to a level of 25%
 by Compounds 2 and 6 and to a level of 37.5% by Compound 1). Retinal edema
 was reduced to 12.5% by Compound 6, to 37.5% by Compound 1 and to 50% by
 Compound 2.
 EXAMPLE 4
 An additional study was conducted. This study was based on the suggestion
 that certain cytokines play a role in the neovascularization process with
 the concentration of these cytokines being abnormal when the undesirable
 neovascularization takes place. An effective agent would correct these
 abnormalities. In one study, the concentration of the cytokine, vascular
 endothelial growth factor ("VEGF") was studied. VEGF concentration was
 measured by immunoassay (R&D Systems Quantkine kit). Measurements were
 made in control animals, control animals receiving an injection of LHP and
 test animals receiving LHP plus test compound. Measurements were carried
 out at the injection site and in the superior quadrant. LHP stimulated the
 maximum synthesis of VEGF between 6 to 24 hours.
 a. Cornea
 Since both areas (injection site and superior quadrant) were decreased in
 treated animals, the values were added together and averaged. The degree
 of reduction produced by NRT compounds at 12 hours post-injection of LHP
 ranged from 55% for Compound 6, to 48% for Compound 1 to 40% for Compound
 2. The concentration of VEGF declined further to 50% to 75% levels at 7
 days and 14 days. These results are presented graphically in FIG. 5.
 b. Retina
 VEGF was reduced by NRT compounds to a greater extent than observed in
 cornea. At 12 hours, 7 or 14 days, the difference was 30% greater in the
 retina (FIG. 6). By 14 days post-injection, Compound 1 inhibited VEGF
 production 92%, Compound 2 inhibited 86% and Compound 6 inhibited 76%.
 This placed all 3 drugs within the range of control samples. These results
 are presented graphically in FIG. 6.
 c. Tumor necrosis factor alpha (TNF.alpha.) addition
 Measurements of tumor necrosis factor alpha (TNF.alpha.) were added to the
 protocol to obtain a more comprehensive understanding of alterations
 occurring in the initiation of the angiogenic cytokine cascade. Tissue
 levels were quantified using a WEH1 cell bioassay which is specific for
 TNF.alpha..
 Previous studies in our laboratory have demonstrated that during the first
 day after LHP exposure, there is a dramatic increase in TNF.alpha. and if
 inhibitors (anti-TNF.alpha. or pentoxifylline) are added in vivo,
 neovascularization is markedly retarded. In the cornea, Compound 2
 depressed TNF.alpha. levels at 12 hours by 36% and at 7 days was still 25%
 below LHP control levels. (These results are shown in FIG. 7) Corneal
 samples at 12 hours from Compound 1 and Compound 6 were contaminated.
 Compound 1- and Compound 6-treated rabbits had TNF.alpha. levels that were
 increased above the baseline at 7 and 14 days post-injection.
 In the retina, Compounds 1, 2 and 6 all inhibited TNF.alpha. synthesis,
 with Compound 6 showing the greatest effect at 12 hours post-injection.
 Compound 2 and Compound 1 appeared to stimulate new synthesis, at 7 days
 after exposure, and then dropped to low levels, whereas Compound 6
 remained near baseline over the 14 days experimental period. (These
 results are presented in FIG. 8)
 While early response of TNF.alpha. provides localized signals for synthesis
 of other cytokines to sustain growth and can be considered a pathological
 event, the increases at 7 days in retina and 14 days in cornea may
 represent secondary repair process. For example, TNF.alpha. may be
 cytotoxic in one situation and restorative in another. Therefore, repair
 stimuli may be regulated differently than the initial oxidative stress
 which initiated neovascularization from the parent vessel.
 In summary, in this study all three NRT compounds tested showed an
 inhibitory effect on LHP induced neovascularization in both cornea and
 retina.
 As a model for studying diabetic retinopathy, the effect of NRTs in
 protecting against induction and associated pathophysiologic changes by
 LHP was important. NRT compounds were observed to affect the synthesis of
 both TNF.alpha. and VEGF which are essential growth factors for the
 initiation and propagation of new vessels. The collective reduction of
 these cytokines would be expected to abate the neovascular responses.
 Retinal neovascular proliferation was reduced best (88%) by Compound 2
 with the other two drugs ranging from 63% to 75%. Compound 6 appeared to
 be more effective against controlling edema, hemorrhage and retinal
 detachment. These results suggest the efficacy of using NRT compounds in
 the management of proliferative diabetic retinopathy.
 Using a more easily visualized corneal model, a marked inhibitory effect
 was also observed. This, too, was correlated with statistically
 significant reductions in the cytokine growth factors TNF.alpha. and VEGF.