Patent Grant
9855239
A diminished visual acuity or total loss of vision may result from a number of eye diseases or disorders caused by dysfunction of tissues or structures in the anterior segment of the eye and/or posterior segment of the eye. To understand why human vision declines with age, much research has focused on the retina, the layer of rod and cone photoreceptors cells that convert light into electrical signals. Studies in mice have shown that age-related decreases in retinal rod cell function cannot be explained by rod cell loss, abnormal retinal plasticity or any signs of retinal disease (Jackson, G. R., Owsley, C. & McGwin, G., Jr., Vision Research, 39, 3975-3982 (1999); Gao, H. & Hollyfield, J. G., Investigative Ophthalmology & Visual Science, 33, 1-17 (1992); Jackson, G. R., Owsley, C., Cordle, E. P. & Finley, C. D. Vision Research, 38, 3655-3662 (1998)). Indeed, Jackson and colleagues reported a dramatic slowing of rod-mediated dark adaptation after light exposure associated with human aging that was related to delayed regeneration of rhodopsin (Jackson, G. R., Owsley, C. & McGwin, G., Jr., Vision Research, 39, 3975-3982 (1999)).
Age related macular degeneration (AMD) is one of the specific diseases associated with the posterior portion of the eyeball and is the leading cause of blindness among older people. AMD results in damage to the macula, a small circular area in the center of the retina. Because the macula is the area which enables one to discern small details and to read or drive, its deterioration may bring about diminished visual acuity and even blindness. The retina contains two forms of light receiving cells, rods and cones, that change light into electrical signals. The brain then converts these signals into the images. The macula is rich in cone cells, which provides central vision. People with AMD suffer deterioration of central vision but usually retain peripheral sight.
Inadequate availability and/or processing of vitamin A to the visual chromophore, 11-cis-retinal, can adversely affect vertebrate rhodopsin regeneration and visual transduction (reviewed in McBee, J. K., Palczewski, K., Baehr, W. & Pepperberg, D. R., Prog Retin Eye Res, 20, 469-529 (2001); Lamb, T. D. & Pugh, E. N., Jr., Prog Retin Eye Res, 23, 307-380 (2004); and Travis, G. H., Golczak, M., Moise, A. R. & Palczewski, K., Annu Rev Pharmacol Toxicol (2006). As in aging, rhodopsin regeneration after light exposure is more delayed in humans and mice deprived of vitamin A due to either dietary deficiency or inadequate intestinal absorption (Lamb, T. D. & Pugh, E. N., Jr., Prog Retin Eye Res, 23, 307-380 (2004)). Moreover, treatment with vitamin A and its derivatives may have beneficial effects in aging (Jacobson, S. G., et al., Nat Genet, 11, 27-32 (1995)) and retinal diseases such as Sorbsby's fundus dystrophy (Jacobson, S. G., et al., Nat Genet, 11, 27-32 (1995)) and retinitis pigmentosa (Berson, E. L., et al., Arch Ophthalmol, 111, 761-772 (1993)).
Retinoid absorption, storage and recycling after bleaching of retinal pigments is impaired in mice lacking lecithin:retinol acyltransferase (LRAT) (Imanishi, Y., Batten, M. L., Piston, D. W., Baehr, W. & Palczewski, K., J Cell Biol 164, 373-383 (2004); Batten, M. L., et al., PLoS Medicine, 2, e333 (2005); Batten, M. L., et al., J Biol Chem, 279, 10422-10432 (2004); O'Byrne, S. M., et al., J Biol Chem, 280, 35647-35657 (2005)) and a null mutation in the human LRAT gene results in early-onset rod-cone dystrophy (Thompson, D. A., et al., Nat Genet, 28, 123-124 (2001)). The latter resembles a form of human Leber's congenital amaurosis (LCA) in which disabling mutations in the retinal pigment epithelium-specific 65 kDa (RPE65) gene also cause severe rod and cone photoreceptor dysfunction (Thompson, D. A., et al., Nat Genet, 28, 123-124 (2001)). LCA patients carrying mutations in both the LRAT and RPE65 genes may, like Lrat−/− and Rpe65−/− knockout mice, lack 11-cis-retinal and rhodopsin, possess abnormalities in all-trans-retinyl ester levels within RPE cells, show severe impairment of rod and cone photoreceptor functions and exhibit retinal degeneration (Imanishi, Y., Batten, M. L., Piston, D. W., Baehr, W. & Palczewski, K., J Cell Biol, 164, 373-383 (2004); Batten, M. L., et al. PLoS Medicine, 2, e333 (2005); Redmond, T. M., et al., Nat Genet, 20, 344-351 (1998); Van Hooser, J. P., et al., Proceedings of the National Academy of Sciences of the United States of America, 97, 8623-8628 (2000); Van Hooser, J. P., et al., J Biol Chem, 277, 19173-19182 (2002)).
The biochemical defects causing LCA-like symptoms in Lrat−/− and Rpe65−/− knockout mice can be bypassed by oral gavage with 9-cis-retinal. This treatment results in preserved retinal morphology and recovery of normal rod function as assessed by single cell recordings and ERG measurements (Batten, M. L., et al., PLoS Medicine, 2, e333 (2005); Van Hooser, J. P., et al., Proceedings of the National Academy of Sciences of the United States of America, 97, 8623-8628 (2000); Van Hooser, J. P., et al., J Biol Chem, 277, 19173-19182 (2002)). 9-cis-retinal forms photoactive isorhodopsin which, when bleached, undergoes conformational changes via the same photoproducts as does rhodopsin naturally regenerated from 11-cis-retinal (Yoshizawa, T. & Wald, G., Nature, 214, 566-571 (1967)). In addition, 11-cis-retinal given by intraperitoneal injection also improves vision in Rpe65−/− mice (Ablonczy, Z., et al., J Biol Chem, 277, 40491-40498 (2002)). Further, gastric gavage with a more chemically stable compound than either 9-cis- or 11-cis-retinal, i.e. 9-cis-retinyl acetate (9-cis-R-Ac), produces the same beneficial effects as 9-cis-retinal in Lrat−/− mice (Batten, M. L., et al., PLoS Medicine, 2, e333 (2005)). Other synthetic retinal derivatives that can be used to restore and/or stabilize photoreceptor function have been described, for example, in International Patent Publication No. WO 2006/002097 A2.
Currently, there are few treatments for retinoid deficiency. One treatment, a combination of antioxidant vitamins and zinc, produces only a small restorative effect by slowing the progression of AMD. Thus, there is a need for methods of restoring or stabilizing photoreceptor function in aging subjects. The present invention is related to the surprising discovery that long-term treatment with a synthetic retinoid derivative significantly improves age-related deterioration of photoreceptor function.
The present invention provides methods of treating or preventing age-related visual impairment comprising long-term administration of one or more synthetic retinal derivatives.
In one embodiment, the present invention provides a method of treating or preventing age-related retinal dysfunction in a subject comprising administering to the subject a pharmaceutically effective amount of a synthetic retinal derivative, wherein the synthetic retinal derivative is administered to the subject for a period of at least three months.
In one embodiment, the synthetic retinal derivative is administered to the subject about once every two weeks to about once every six weeks for a period of at least three months.
In another embodiment, the retinal derivative is administered to the subject about once a month for a period of from about 6 to about 10 months.
In another embodiment, the age-related retinal dysfunction is manifested by one or more of the following clinical conditions: an impairment in rod-mediated dark adaptation after light exposure, an impairment in night vision, an impairment in contrast sensitivity, and age-related macular degeneration (AMD).
In yet another embodiment, the present invention provides a method of improving rhodopsin regeneration ratio in a mammal comprising administering to the mammal a pharmaceutically effective amount of a synthetic retinal derivative, wherein the synthetic retinal derivative is administered to the mammal for a period of at least three months.
The present methods are directed to treating or preventing age-related retinal dysfunction in a subject via long-term administration of a pharmaceutically effective amount of a synthetic retinal derivative.
As used herein, the term “age-related retinal dysfunction” refers to age-related decreases in retinal photoreceptor function. The term is meant to include the age-related impairments related to electroretinogram deficits and photoreceptor cell death and structural abnormalities that have been observed in both animal and human studies of aging. In one aspect, the age-related retinal dysfunction comprises a slowing of rod-mediated dark adaptation after light exposure, a decrease in night vision, and/or a decrease in contrast sensitivity. In another aspect, the age-related retinal dysfunction comprises age-related macular degeneration (AMD). The AMD can be wet or dry forms.
The terms “treating,” treatment,” and the like are used herein to generally mean obtaining a desired pharmacological and physiological effect. More specifically, the synthetic retinal derivatives described herein which are used to treat a subject with age-related retinal dysfunction generally are provided in a therapeutically effective amount to achieve an improvement in age-related retinal dysfunction or an inhibited development of age-related retinal dysfunction in the visual system of an ageing subject, as compared with a comparable visual system not receiving the synthetic retinal derivative. An improvement in age-related retinal dysfunction includes long-term (e.g., as measured in weeks or months) improvement or restoration of photoreceptor function in a visual system, as compared with a comparable visual system not receiving the synthetic retinal derivative. Improvement also includes stabilization of, or minimization of additional degradation in, a vertebrate visual system, as compared with a comparable vertebrate visual system not receiving the synthetic retinal derivative.
The terms “preventing,” “prevention,” and the like are used generally to mean preventing or inhibiting deterioration or further deterioration of the visual system of an aging subject, as compared with a comparable visual system not receiving the synthetic retinal derivative.
The term “pharmaceutically effective” as used herein refers to the effectiveness of a particular treatment or prevention regime. Pharmaceutical efficacy can be measured based on such characteristics as, for example, an increased or stabilized rate of dark-adaptation, a higher or stabilized rhodpsin/opsin ratio, a higher or stabilized rhodopsin regeneration rate, or other such improvements in electrotretinographic (ERG) responses.
In the present methods, a synthetic retinal derivative is administered to a subject. As used herein, the term “subject” or “patient” refers to a vertebrate, for example a mammal such as a human. In one embodiment, the subject is an aging subject, such as a human, suffering from age-related retinal dysfunction. As used herein, an aging human subject is typically at least 45, or at least 50, or at least 60, or at least 65 years old. The subject has an aging eye, which is characterized as having age-related retinal dysfunction. Age-related retinal dysfunction may be manifested by one or more of the following clinical conditions: an impairment in rod-mediated dark adaptation after light exposure, an impairment in night vision, an impairment in contrast sensitivity, and age-related macular degeneration (AMD).
The synthetic retinal derivative is administered using long-term (chronic) dosage regimens. In one embodiment, the synthetic retinal derivative is administered intermittently for three months or longer; and, in another embodiment, for six months or longer. The synthetic retinal derivative can be administered, for example, for a period of about three, four, five, six, seven, eight, nine, ten, eleven, or twelve months, or longer. The synthetic retinal derivative can be intermittently administered to the subject about once a day to about once every two months. Intermittent administration includes administration to the subject about once every other day; about four times a week, three times a week, and two times a week; about once every two, three, four, five, six, seven, eight, and nine weeks; and about once a month. In one embodiment, the synthetic retinal derivative is administered about once every three to six weeks for a period of about three months or longer; and in another embodiment, it is administered about once a month for about six to ten months.
The amount of synthetic retinal derivative administered per dose can be increased as the time period between doses is increased. For example, if the synthetic retinal derivative is administered less than once a day, the dose per administration can be greater than the effective daily dose. As used herein, an “effective daily dose” refers to a daily dose effective for obtaining a desired pharmacological and physiological effect (i.e. a daily dose effective for “treating” and/or “preventing” age related retinal dysfunction in a subject as described above).
In addition, the synthetic retinal derivative can be chronically released from a controlled drug delivery formulation and/or device for an extended period of time, e.g., for a period of about three months or longer; or for a period of about six months or longer. A wide variety of methods for controlled release have been developed and are known to those skilled in the art, including pumps, patches, tablets, implants, microchips, and polymeric systems.
Suitable doses of synthetic retinal derivatives will depend on the clinical status, condition and age of the patient, the active agent, the formulation and dosage form, the frequency of dosing, and the like. In many instances the selection of an appropriate dose will be within the skill of a suitable healthcare practitioner such as a physician or nurse. In the case of eye drops, a synthetic retinal derivative can be administered, for example, from about 0.01 mg, about 0.1 mg, or about 1 mg, to about 25 mg, to about 50 mg, or to about 90 mg per single dose. In the case of injection, suitable doses are about 0.0001 mg, about 0.001 mg, about 0.01 mg, or about 0.1 mg to about 10 mg, to about 25 mg, to about 50 mg, or to about 500 mg of the synthetic retinal derivative. Suitable oral doses range from about 0.1 to about 1000 mg of the synthetic retinal derivative. In other embodiments, about 1.0 to about 300 mg of synthetic retinal derivative can be administered per dose.
In certain embodiments, the dose is an oral dose of about 0.01 to about 10 mg/kg body weight; about 0.05 to about 7.5 mg/kg body weight; about 0.1 to about 5 mg/kg body weight; or about 0.5 to about 2.5 mg/kg body weight. For example, the synthetic retinal derivative can be administered at an oral dosage of about 6.4 mg/kg body weight (i.e. about 240 mg/m2 body surface area). In another embodiment, the dose is an oral daily dose of about 0.1 to about 1 mg/kg body weight, such as an oral daily dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/kg body weight.
Synthetic retinal derivatives suitable for the methods of the present invention have been described in International Patent Publication Nos. WO 2004/082622 A2, published in the U.S. as US 2004/0242704, and WO 2006/002097 A2, published in the U.S. as US 2008/0221208 and in U.S. Patent Publication No. 2004/0242704A1.
A synthetic retinal derivative suitable for the methods of the present invention is a derivative of 9-cis-retinal or 11-cis-retinal in which the aldehydic group in the polyene chain is modified. The synthetic retinal derivative can be converted directly or indirectly into a retinal or a synthetic retinal analog. Thus, in some aspects, the compounds according to the present invention can be described as pro-drugs, which upon metabolic transformation are converted into 9-cis-retinal, 11-cis-retinal or a synthetic retinal analog thereof. Metabolic transformation can occur, for example, by acid hydrolysis, esterase activity, acetyltransferase activity, dehydrogenase activity, or the like.
The synthetic retinal derivative can be a retinoid replacement, supplementing the levels of endogenous retinoid. In some embodiments, the synthetic retinal can bind to opsin, and function as an opsin agonist. As used herein, the term “agonist” refers to a synthetic retinal that binds to opsin and facilitates the ability of an opsin/synthetic retinal complex to respond to light. As an opsin agonist, a synthetic retinal can spare the requirement for endogenous retinoid (e.g., 11-cis-retinal). A synthetic retinal also can restore or improve function (e.g., photoreception) to opsin by binding to opsin and forming a functional opsin/synthetic retinal complex, whereby the opsin/synthetic retinal complex can respond to photons when part of a rod or cone membrane.
Synthetic retinal derivatives can be administered to restore or stabilize photoreceptor function, and/or to ameliorate the effects of a deficiency in retinoid levels. Photoreceptor function can be restored or stabilized, for example, by providing a synthetic retinal derivative as an 11-cis-retinoid replacement and/or an opsin agonist. The synthetic retinal derivative also can ameliorate the effects of a retinoid deficiency on a vertebrate visual system. The synthetic retinal derivative can be administered prophylactically or therapeutically to a vertebrate. Suitable vertebrates include, for example, human and non-human vertebrates. Suitable non-human vertebrates include, for example, mammals, such as dogs (canine), cats (feline), horses (equine) and other domesticated animals.
In one aspect of the invention, the synthetic retinal derivatives are derivatives of 9-cis-retinal or 11-cis-retinal in which the aldehydic group in the polyene chain is converted to an ester, ether, alcohol, hemi-acetal, acetal, or oxime, as further described herein. Such synthetic retinal derivatives include 9-cis-retinyl esters, 9-cis-retinyl ethers, 9-cis-retinol, 9-cis retinyl oximes, 9-cis-retinyl acetals, 9-cis-retinyl hemiacetals, 11-cis-retinyl esters, 11-cis-retinyl ethers, 11-cis-retinol, 11-cis-retinyl oximes, 11-cis-retinyl acetals and II-cis-retinyl hemiacetals, as further described herein. The synthetic retinal derivative can be metabolized to release a natural or synthetic retinal, such as for example, 9-cis-retinal, 11-cis-retinal or a synthetic retinal analog thereof, such as those described herein or in International Patent Publication Nos. WO 2004/082622 A2, published in the U.S. as US 2004/0242704, and WO 2006/002097 A2, published in the U.S. as US 2008/0221208.
In one aspect, the synthetic retinal derivative is a retinyl ester. In some embodiments, the retinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl ester having a. The ester substituent can be, for example, a carboxylic acid, such as a mono- or polycarboxylic acid. As used herein, a “polycarboxylic acid” is a di-, tri- or higher order carboxylic acid. In some embodiments, the carboxylic acid is a C1-C22, C2-C22, C3-C22, C1-C10, C2-C10, C3-C10, C4-C10, C4-C8, C4-C6 or C4 monocarboxylic acid, or polycarboxylic acid.
Suitable carboxylic acid groups include, for example, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, oleic acid, stearic acid, palmitic acid, myristic acid or linoleic acid. The carboxylic acid also can be, for example, oxalic acid (ethanedioic acid), malonic acid (propanedioic acid), succinic acid (butanedioic), fumaric acid (butenedioic acid), malic acid (2-hydroxybutenedioic acid), glutaric acid (pentanedioic acid), adipic acid (hexanedioic acid), pimelic acid (heptanedioic), suberic acid (octanedioic), azelaic acid (nonanedioic acid), sebacic acid (decanedioic acid), citric acid, oxaloacetic acid, ketoglutaratic acid, or the like.
In an exemplary embodiment, the retinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl ester including a C3-C10 polycarboxylic acid substituent. (In this context, the terms “substituent” or “group” refer to a radical covalently linked to the terminal oxygen in the polyene chain.) In another exemplary embodiment, the retinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl ester including a C2-C22 or C3-C22 polycarboxylic acid substituent. The polycarboxylic acid substituent can be, for example, succinate, citrate, ketoglutarate, fumarate, malate or oxaloacetate. In another exemplary embodiment, the retinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl ester including a C3-C22 di-carboxylic acid (di-acid) substituent. In some embodiments, the polycarboxylic acid is not 9-cis-retinyl tartarate or 11-cis-retinyl tartarate. In some embodiments, the retinyl ester is not a naturally occurring retinyl ester normally found in the eye. In some embodiments, the retinyl ester is an isolated retinyl ester. As used herein, “isolated” refers to a molecule that exists apart from its native environment and is therefore not a product of nature. An isolated molecule may exist in a purified form or may exist in a non-native environment.
In another aspect, the retinal derivative can be a 9-cis-retinyl ester or ether of the following formula I:
In some embodiments, A is CH2OR, where R can be an aldehydic group, to form a retinyl ester. A suitable aldehydic group is a C1 to C24 straight chain or branched aldehydic group. The aldehydic group also can be a C1 to C14 straight chain or branched aldehydic group. The aldehydic group can be a C1 to C12 straight chain or branched aldehydic group, such as, for example, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal. R can be a C1 to C10 straight chain or branched aldehydic group, a C1 to C8 straight chain or branched aldehydic group or a C1 to C6 straight chain or branched aldehydic group.
R further can be a carboxylate group of a dicarboxylic acid or other carboxylic acid (e.g., a hydroxyl acid) to form a retinyl ester (some of which are also referred to as retinoyl esters). The carboxylic acid can be, for example, oxalic acid (ethanedioic acid), malonic acid (propanedioic acid), succinic acid (butanedioic), fumaric acid (butenedioic acid), malic acid (2-hydroxybutenedioic acid), glutaric acid (pentanedioic acid), adipic acid (hexanedioic acid), pimelic acid (heptanedioic), suberic acid (octanedioic), azelaic acid (nonanedioic acid), sebacic acid (decanedioic acid), citric acid, oxaloacetic acid, ketoglutaratic acid, or the like.
R can also be an alkane group, to form a retinyl alkane ether. Suitable alkane groups include, for example, C1 to C24 straight chain or branched alkyls, such as, for example, methane, ethane, butane, isobutane, pentane, isopentane, hexane, heptane, octane or the like. For example, the alkane group can be a linear, iso-, sec-, tert- or other branched lower alkyl ranging from C1 to C6. The alkane group also can be a linear, iso-, sec-, tert- or other branched medium chain length alkyl ranging from C8 to C14. The alkane group also can be a linear, iso-, sec-, tert- or other branched long chain length alkyl ranging from C16 to C24.
R further can be an alcohol group, to form a retinyl alcohol ether. Suitable alcohol groups can be linear, iso-, sec-, tert- or other branched lower alcohols ranging from C1 to C6, linear, iso-, sec-, tert- or other branched medium chain length alcohols ranging from C8 to C14, or linear, iso-, sec-, tert- or other branched long chain length alkyl ranging from C16 to C24. The alcohol group can be, for example, methanol, ethanol, butanol, isobutanol, pentanol, hexanol, heptanol, octanol, or the like
R also can be a carboxylic acid, to form a retinyl carboxylic acid ether. Suitable alcohol groups can be linear, iso-, sec-, ted- or other branched lower carboxylic acids ranging from C1 to C6, linear, iso-, sec-, ted- or other branched medium chain length carboxylic acids ranging from C8 to C14, or linear, iso-, sec-, tert- or other branched long chain length carboxylic acids ranging from C16 to C24. Suitable carboxylic acid groups include, for example, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, oleic acid, stearic acid, palmitic acid, myristic acid, linoleic acid, succinic acid, fumaric acid or the like.
The retinyl derivative can be a retinyl hemiacetal, where A is CH(OH)OR. R can be any of the R groups set forth above in Formula I. R is typically a lower alkane, such as a methyl or ethyl group, or a C1 to C7 saturated and unsaturated, cyclic or acyclic alkane, with or without hetero atoms, as described herein.
The retinyl derivative can be a retinyl acetal, where A is CH (ORa)ORb. Each of Ra and Rb can be independently selected from any of the R groups set forth above in Formula I. Ra and Rb are typically a C1 to C7 saturated and unsaturated, cyclic or acyclic alkane, with or without hetero atoms, as described herein.
The retinyl derivative also can be a retinyl oxime, where A is CH:NOH or CH:NOR. R can be any of the R groups set forth above in Formula I. R is typically a hydrogen, or an alkane.
Examples of suitable synthetic retinal derivatives include, for example, 9-cis-retinyl acetate, 9-cis-retinyl formate, 9-cis-retinyl succinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate, 9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyl oxaloacetate, 9-cis-retinal oxime, 9-cis-retinal O-methyl oximes, 9-cis-retinal O-ethyl oximes, and 9-cis-retinal methyl acetals and hemi acetals, 9-cis-retinyl methyl ether, 9-cis-retinyl ethyl ether, and 9-cis-retinyl phenyl ether.
In a related aspect, the retinal derivative can be an 11-cis-retinyl ester or ether of the following formula II:
A can be any of the groups set forth above in Formula I.
Examples of suitable synthetic retinal derivatives include, for example, 11-cis-retinyl acetate, 11-cis-retinyl formate, 11-cis-retinyl succinate, 11-cis-retinyl citrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate, 11-cis-retinyl malate, 11-cis-retinal oxime,11-cis-retinal O-methyl oxime, 11-cis-retinal O-ethyl oximes and 11-cis-retinal methyl acetals and hemi acetals, 11-cis-retinyl methyl ether, 11-cis-retinyl ethyl ether.
In additional aspects, the synthetic retinal derivatives can be, for example, a derivative of a 9-cis-retinyl ester, a 9-cis-retinyl ether, an 11-cis-retinyl ester or an 11-cis-retinyl ethers such as, for example, an acyclic retinyl ester or ethers, a retinyl ester or ether with a modified polyene chain length, such as a trienoic or tetraenoic retinyl ester or ether; a retinyl ester or ether with a substituted polyene chain, such as alkyl, halogen or heteratom-substituted polyene chains; a retinyl ester or ether with a modified polyene chain, such as a trans- or cis-locked polyene chain, or with, for example, allene or alkyne modifications; and a retinyl ester or ether with a ring modification(s), such as heterocyclic, heteroaromatic or substituted cycloalkane or cycloalkene rings.
The synthetic retinal derivative can be a retinyl ester or ether of the following formula III:
A can be any of the groups set forth above for formula (I). R1 and R2 can be independently selected from linear, iso-, sec-, tert- and other branched alkyl groups as well as substituted alkyl groups, substituted branched alkyl, hydroxyl, hydroalkyl, amine, amide, or the like. R1 and R2 can independently be lower alkyl, which means straight or branched alkyl with 1-6 carbon atom(s) such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, or the like. Suitable substituted alkyls and substituted branch alkyls include, for example, alkyls, branched alkyls and cyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatom or other groups. Suitable heteroatoms include, for example, sulfur, silicon, and fluoro- or bromo-substitutions.
R1 or R2 also can be a cyclo-alkyl such as, for example, hexane, cyclohexene, benzene as well as a substituted cyclo-alkyl. Suitable substituted cyclo-alkyls include, for example, cyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatom and/or other groups. Suitable heteroatoms include, for example, sulfur, silicon, and fluoro- or bromo-substitutions.
The synthetic retinal derivative also can have a modified polyene chain length, such as the following formula IV:
A can be any of the groups set forth above for formula (I). The polyene chain length can be extended by 1, 2, or 3 alkyl, alkene or alkylene groups. According to formula (IV), each n and n1 can be independently selected from 1, 2, or 3 alkyl, alkene or alkylene groups, with the proviso that the sum of the n and n1 is at least 1.
The synthetic retinal derivative also can have a substituted polyene chain of the following formula V:
A can be any of the groups set forth above for formula (I). Each of R1 to R8 can be independently selected from hydrogen, alkyl, branched alkyl, cyclo-alkyl, halogen, a heteratom, or the like. Suitable alkyls include, for example, methyl, ethyl, propyl, substituted alkyl (e.g., alkyl with hydroxyl, hydroalkyl, amine, amide) or the like. Suitable branched alkyls can be, for example, isopropyl, isobutyl, substituted branched alkyl, or the like. Suitable cyclo-alkyls can include, for example, cyclohexane, cycloheptane, and other cyclic alkanes as well as substituted cyclic alkanes such as substituted cyclohexane or substituted cycloheptane. Suitable halogens include, for example, bromine, chlorine, fluorine, or the like. Suitable heteroatoms include, for example, sulfur, silicon, and fluoro- or bromo-substitutions. Suitable substituted alkyls, substituted branch alkyls and substituted cyclo-alkyls include, for example, alkyls, branched alkyls and cyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatom or other groups.
For example, the synthetic retinal derivative can be selected from the following: a 9-ethyl-11-cis-retinyl ester, ether, oxime, acetal or hemiacetal; a 7-methyl-11-cis-retinyl ester, ether, oxime, acetal or hemiacetal; a 13-desmethyl-11-cis-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-10-F-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-10-Cl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-10-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-10-ethyl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-10-F-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-10-Cl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-10-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-10-ethyl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-12-F-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-12-Cl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-12-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-10-ethyl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-12-F-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-12-Cl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-12-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-14-F-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-14-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; an 11-cis-14-ethyl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-14-F-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-14-methyl-retinyl ester, ether, oxime, acetal or hemiacetal; a 9-cis-14-ethyl-retinyl ester, ether, oxime, acetal or hemiacetal; or the like.
The synthetic retinal derivative further can have a modified ring structure. Suitable examples include, for example, derivatives containing ring modifications, aromatic analogs and heteroaromatic analogs of the following formulae VI, VII and VIII, respectively:
A can be any of the groups set forth above for formula (I). Each of R1 to R6, as applicable, can be independently selected from hydrogen, alkyl, substituted alkyl, hydroxyl, hydroalkyl, amine, amide, halogen, a heteratom, or the like. Suitable alkyls include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or the like. Suitable halogens include, for example, bromine, chlorine, fluorine, or the like. Suitable heteroatoms include, for example, sulfur, silicon, or nitrogen. In formulae VII, X can be, for example, sulfur, silicon, nitrogen, fluoro- or bromo-substitutions. Similarly, 9-cis-synthetic retinal derivatives containing ring modifications, aromatic analogs and heteroaromatic analogs of those shown in formulae VI, VII and VIII are contemplated.
The synthetic retinal derivative also can have a modified polyene chain. Suitable derivatives include, for example, those with a trans/cis locked configuration, 6s-locked analogs, as well as modified allene, alkene, alkyne or alkylene groups in the polyene chain. In one example, the derivative is an 11-cis-locked analog of the following formula IX:
A can be any of the groups set forth above for formula (I). R3 can be, for example, hydrogen, methyl or other lower alkane or branch alkane. n can be 0 to 4. m plus 1 equals 1, 2 or 3.
In one embodiment, the synthetic retinal derivative can be an 11-cis-locked analog of the following formula X:
n can be 1 to 4. A can be any of the groups set forth above for formula (I).
The synthetic retinal derivative is a 9,11,13-tri-cis-7-ring retinyl ester or ether, an 11,13-di-cis-7-ring retinyl ester or ether, an 11-cis-7-ring retinyl ester or ether or a 9,11-di-cis-7-ring retinyl ester or ether.
In another example, the synthetic retinal derivative is a 6s-locked analog of formula XI. A can be any of the groups set forth above for formula (I). R1 and R2 can be independently selected from hydrogen, methyl and other lower alkyl and substituted lower alkyl. R3 can be independently selected from an alkene group at either of the indicated positions.
The synthetic retinal derivative can be a 9-cis-ring-fused derivative, such as, for example, those shown in formulae XII-XIV. A can be any of the groups set forth above for formula (I).
The synthetic retinal derivative also can be of the following formula XV or XVI.
A can be any of the groups set forth above for formula (I). Each of R2 to R5, R7 to R14, R16 and R17 can be absent or independently selected from hydrogen, alkyl, branched alkyl, halogen, hydroxyl, hydroalkyl, amine, amide, a heteratom, or the like. Suitable alkyls include, for example, methyl, ethyl, propyl, substituted alkyl (e.g., alkyl with hydroxyl, hydroalkyl, amine, amide), or the like. Suitable branched alkyl can be, for example, isopropyl, isobutyl, substituted branched alkyl, or the like. Suitable halogens include, for example, bromine, chlorine, fluorine, or the like. Suitable heteroatoms include, for example, sulfur, silicon, and fluoro- or bromo-substitutions. Suitable substituted alkyls and substituted branch alkyls include, for example, alkyls and branched alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatom or other groups. Each of n and n1 can be independently selected from 1, 2, or 3 alkyl, alkene or alkylene groups, with the proviso that the sum of the n and n1 is at least 1. In addition, R3-R4 and/or R2-R16 can comprise an alkene group in the cyclic carbon ring, in which case R17 is absent. R10 and R13 together can form a cyclo-alkyl, such as a five, six, seven or eight member cyclo-alkyl or substituted cyclo-alkyl, such as, for example, those shown in Formulae IX, X, XII, XIII and XIV.
Methods of making synthetic retinals and derivatives are disclosed in, for example, the following references: Anal. Biochem. 272:232-42 (1999); Angew. Chem. 36:2089-93 (1997); Biochemistry 14:3933-41 (1975); Biochemistry 21:384-93 (1982); Biochemistry 28:2732-39 (1989); Biochemistry 33:408-16 (1994); Biochemistry 35:6257-62 (1996); Bioorganic Chemistry 27:372-82 (1999); Biophys. Chem. 56:31-39 (1995); Biophys. J. 56:1259-65 (1989); Biophys. J. 83:3460-69 (2002); Chemistry 7:4198-204 (2001); Chemistry (Europe) 5:1172-75 (1999); FEBS 158:1 (1983); J. Am. Chem. Soc. 104:3214-16 (1982); J. Am. Chem. Soc. 108:6077-78 (1986); J. Am. Chem. Soc. 109:6163 (1987); J. Am. Chem. Soc. 112:7779-82 (1990); J. Am. Chem. Soc. 119:5758-59 (1997); J. Am. Chem. Soc. 121:5803-04 (1999); J. American Chem. Soc. 123:10024-29 (2001); J. American Chem. Soc. 124:7294-302 (2002); J. Biol. Chem. 276:26148-53 (2001); J. Biol. Chem. 277:42315-24 (2004); J. Chem. Soc.-Perkin T. 1:1773-77 (1997); J. Chem. Soc-Perkin T. 1:2430-39 (2001); J. Org. Chem. 49:649-52 (1984); J. Org. Chem. 58:3533-37 (1993); J. Physical Chemistry B 102:2787-806 (1998); Lipids 8:558-65; Photochem. Photobiol. 13:259-83 (1986); Photochem. Photobiol. 44:803-07 (1986); Photochem. Photobiol. 54:969-76 (1991); Photochem. Photobiol. 60:64-68 (1994); Photochem. Photobiol. 65:1047-55 (1991); Photochem. Photobiol. 70:111-15 (2002); Photochem. Photobiol. 76:606-615 (2002); Proc. Natl Acad. Sci. USA 88:9412-16 (1991); Proc. Natl Acad. Sci. USA 90:4072-76 (1993); Proc. Natl Acad. Sci. USA 94:13442-47 (1997); and Proc. R. Soc. Lond. Series B, Biol. Sci. 233(1270): 55-76 1988) (the disclosures of which are incorporated by reference herein).
Retinyl esters can be formed by methods known in the art such as, for example, by acid-catalyzed esterification of a retinol with a carboxylic acid, by reaction of an acyl halide with a retinol, by transesterification of a retinyl ester with a carboxylic acid, by reaction of a primary halide with a carboxylate salt of a retinoic acid, by acid-catalyzed reaction of an anhydride with a retinol, or the like. In an example, retinyl esters can be formed by acid-catalyzed esterification of a retinol with a carboxylic acid, such as, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, oleic acid, stearatic acid, palmitic acid, myristic acid, linoleic acid, succinic acid, fumaric acid or the like. In another example, retinyl esters can be formed by reaction of an acyl halide with a retinol (see, e.g., Van Hooser et al., Proc. Natl. Acad. Sci., USA, 97:8623-28 (2000)). Suitable acyl halides include, for example, acetyl chloride, palmitoyl chloride, or the like.
Retinyl ethers can be formed by methods known in the art, such as for example, reaction of a retinol with a primary alkyl halide.
Trans-retinoids can be isomerized to cis-retinoids by exposure to UV light. For example, all-trans-retinal, all-trans-retinol, all-trans-retinyl ester or all-trans-retinoic acid can be isomerized to 9-cis-retinal, 9-cis-retinol, 9-cis-retinyl ester or 9-cis-retinoic acid, respectively. trans-Retinoids can be isomerized to 9-cis-retinoids by, for example, exposure to a UV light having a wavelength of about 365 nm, and substantially free of shorter wavelengths that cause degradation of cis-retinoids, as further described herein.
Retinyl acetals and hemiacetals can be prepared, for example, by treatment of 9-cis- and 11-cis-retinals with alcohols in the presence of acid catalysts. Water formed during reaction is removed, for example by Al2O3 of a molecular sieve.
Retinyl oximes can be prepared, for example, by reaction of a retinal with hydroxylamine, O-methyl- or O-ethylhydroxyl amine, or the like.
The synthetic retinal derivative can be substantially pure, in that it contains less than about 5% or less than about 1%, or less than about 0.1%, of other retinoids. A combination of synthetic retinal derivatives can be administered.
Synthetic retinal derivatives can be delivered to the eye by any suitable means, including, for example, oral, intravenous, intramuscular or local administration. Modes of local administration can include, for example, eye drops, intraocular injection or periocular injection, or delivery via a controlled release drug delivery formulation and/or device. Periocular injection typically involves injection of the synthetic retinal derivative into the conjunctiva or to the tennon (the fibrous tissue overlying the eye). Intraocular injection typically involves injection of the synthetic retinal derivative into the vitreous. The administration can be non-invasive, such as by eye drops or oral dosage form.
Synthetic retinal derivatives can be formulated, for example, as pharmaceutical compositions for local administration to the eye and/or for intravenous, intramuscular or oral administration.
Synthetic retinal derivatives can be formulated for administration using pharmaceutically acceptable vehicles as well as techniques routinely used in the art. A vehicle can be selected according to the solubility of the synthetic retinal derivative. Suitable pharmaceutical compositions include those that are administrable locally to the eye, such as by eye drops, injection or the like. In the case of eye drops, the formulation can also optionally include, for example, ophthalmologically compatible agents such as isotonizing agents such as sodium chloride, concentrated glycerin, and the like; buffering agents such as sodium phosphate, sodium acetate, and the like; surfactants such as polyoxyethylene sorbitan mono-oleate (also referred to as Polysorbate 80), polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, and the like; stabilization agents such as sodium citrate, sodium edentate, and the like; preservatives such as benzalkonium chloride, parabens, and the like; and other ingredients. Preservatives can be employed, for example, at a level of from about 0.001 to about 1.0% weight/volume. The pH of the formulation is usually within the range acceptable to ophthalmologic formulations, such as within the range of about pH 4 to 8.
Suitable pharmaceutical compositions also include those formulated for injection. For example, the synthetic retinal derivative can be provided in an injection grade saline solution, in the form of an injectable liposome solution, or other carriers or vehicles. Intraocular and periocular injections are known to those skilled in the art and are described in numerous publications including, for example, Ophthalmic Surgery: Principles of Practice, Ed., G. L. Spaeth, W. B. Sanders Co., Philadelphia, Pa., U.S.A., pages 85-87 (1990).
A synthetic retinal derivative also can be administered in a time release formulation and/or device, for example in a composition which includes a slow release polymer. The synthetic retinal derivative can be prepared with a carrier(s) that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are known to those skilled in the art.
Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. Suitable nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g., Remington “Pharmaceutical Sciences”, 17 Ed., Gennaro (ed.), Mack Publishing Co., Easton, Pa. (1985).)
The following examples are provided merely as illustrative of various aspects of the invention and should not be construed to limit the invention in any way.
Introduction
Both single dose and long-term monthly dosage regimens with 9-cis-R-Ac were used to assess the effect of artificial chromophore augmentation on the visual function of mice. The chromophore used was 9-cis-R-Ac, a pro-drug which is metabolized and converted to 9-cis-retinal to form isorhodopsin (Batten, M. L., et al. PLoS medicine 2, e333 (2005)). Twenty mice were employed for the single dose experiments (
Abbreviations
9-cis-R-Ac, 9-cis-retinyl acetate; A2E, N-retinylidene-N-retinyl ethanolamine; ERG, electroretinogram; LRAT, lecithin:retinol acyltransferase; ROS, rod outer segments; RPE, retinal pigment epithelium; RPE65, a RPE-specific 65 kDa protein.
Forty eight hr dark-adapted, 10-month-old mice were gavaged with a single dose (˜80 mg/kg body weight) of 9-cis-R-Ac or control vehicle and exposed to strong illumination for 20 min (500 cd·m−2 that bleached ˜90% rhodopsin). Next, mice were dark-adapted for 16 hr after which various analyses were performed (
For the long-term studies (
To evaluate the effects of rod- and cone-mediated light responses after long-term 9-cis-R-Ac treatments, mice were examined by non-invasive ERG methods. The first set of analyses was done at 4 and 10 months of age. Under scotopic conditions, the amplitudes of a-waves decreased with age, especially at high flash intensities (C1 versus C0 group,
The second set of analyses was done at 14 months of age. No significant differences were found in a- and b-wave amplitudes between control (C2) and treated groups of mice (N2 and N3) under either scotopic of photopic conditions (
Recovery of the ERG response (dark adaptation) following bleach was measured by monitoring the amplitude of a-waves after retinal exposure to intense constant illumination (500 cd·m−2, ˜90% bleached rhodopsin) for 3 min. Recovery of the responses was significantly faster in the 9-cis-R-Ac treated groups compared with the control groups of mice at both 10 months (N1 vs. C1,
In additional control experiments, mice were gavaged with an inactive isomer, all-trans-R-Ac (n=10) for 10 months and evaluated at 14 months age. ERG examination showed no significant difference compared with control (C2) in single flash ERG a- and b-wave analyses or in dark adaptation recovery (n=3). These results showed that ERG effects are specific for 9-cis isomer.
Regeneration ratios (rhodopsin/opsin) and total amounts of purified rhodopsin showed no significant differences between control and treated groups of mice at both 10 and 14 months of age. The amounts of purified protein recovered from the treated groups at 14 months of age (N2 and N3) were significantly lower than these from control mice (C2), whereas the amount was only slightly attenuated in at 10-month-old treated mice (N1 versus C1) (data not shown).
To evaluate the safety of long-term administration of 9-cis-R-Ac, A2E accumulation was measured (
Animals were evaluated weekly for activity and changes in coat and skin appearance. No changes in these parameters were observed during the experimental period aside from these due to natural aging. Body weights of mice evaluated at pre-treatment, 10 months and 14 months showed no significant differences between control (C1-2) and treated groups (N1-3) (data not shown).
Light microscopy revealed no major abnormalities in the retinas of vehicle, 9-cis-R-Ac or all-trans-R-Ac (n=2) treated mice at 10 and 14 months of age and retinas from these two 9-cis-R-Ac treated groups were indistinguishable. Lengths of rod outer segments (ROS) were similar in control and treated groups at 10- and 14 months of age but were significantly decreased as compared to ROS lengths of 4-month-old mice (C0) whose retinal histology is shown in
DNA microarray analyses were used to document possible changes in gene expression profiles after the long-term treatment with 9-cis-R-Ac. Expression levels of mRNA in the eye, liver and kidney were determined and compared between treated (N2) and control (C2) groups using a 37,364 gene array provided by NimbleGen System Inc. In the eye, 9-cis-R-Ac treatment elevated expression of 290 genes by a factor of 2 or more and attenuated expression of more than 1057 genes by a factor of 0.5 or less (
aMice were used after ERG tests for these experiments.
bOne eye from a mouse was used for light microscopy and the other for electron microscopy.
Discussion
As shown in these studies, age-related deterioration of dark adaptation in mice is attenuated by artificial cis-retinoid treatment. This finding is analogous to age-related declines in human vision manifested by the dramatic slowing of rod-mediated dark adaptation directly attributed to delayed rhodopsin regeneration (Jackson, G. R., Owsley, C. & McGwin, G., Jr. Vision Research 39, 3975-3982 (1999)). Two different types of studies were done. Single dose studies revealed that 9-cis-retinoids could enter the eye, and a second set of experiments showed that long-term administration of 9-cis-R-Ac significantly improved the deterioration of retinal function in aging mice.
Single Dose Experiments
These experiments were designed to test whether 9-cis-retinoids enter the eyes of 10-month-old mice and improve visual function. The ERG response measurably declined with age and small but measurable amounts of free opsin was present in these old mice. 9-cis-retinal entered the eye when these treated mice were exposed to intense light and tested 18 hr later. The precursors of 9-cis-retinal, 9-cis-retinyl esters, were easily detectable in the eyes of these mice and the rhodopsin/opsin ratio improved as well.
Long-Term Treatment of Mice with 9-cis-R-Ac
ERG responses to a single light flash were significantly improved in 10-month-old mice that were treated for 6 months with 9-cis-R-Ac as compared with the oil-treated controls (
Long-term treatment (6-10 months) was well tolerated by C57BL/6 female mice. Age-related detectable morphological changes in the retina were observed in both treated and control mice were not affected by treatment. Potential toxic by-products of retinoid treatment, A2E and retinoic acid, did not accumulate in these mice. In this study, 9-cis-R-Ac was incorporated into free-opsin within the retina resulting in increased regeneration ratio of rod pigments. However, the amount of all-trans-retinal induced by physiological light conditions is constant regardless of the amounts of rod pigments and the regeneration ratio. Therefore, it is not surprising that A2E levels were not affected by 9-cis-R-Ac administration. Thus, the beneficial effect of cis-retinoid supplementation seems more advantageous from this perspective than from the anti-oxidant properties generally attributed to retinoids (Maxwell, S. & Greig, L. Expert Opinion on Pharmacotherapy 2, 1737-1750 (2001)).
We did not detect any accumulation of retinoic acids. Moreover, gene expression changes were minimal in the liver and kidney while several proteins were unregulated in the eye. Our detailed analysis of these genes did not reveal any particular expression patterns.
Prophylactic Use of 9-cis-retinoids
Humans begin to lose their ability to dark adapt beginning in the 3rd-4th decade (Jackson, G. R., McGwin, G., Jr., Phillips, J. M., Klein, R. & Owsley, C. Vision Research 46, 1422-1431 (2006)). A decline in the visual function is functionally manifested by reduction in the ability to perform activities such as driving at night and reading in darkened environments (Schilling, O. K. & Wahl, H. W. Psychology and aging 21, 703-714 (2006)). Such symptoms become more debilitating with age and can result in reduced independence and activity in the elderly. The problem becomes more acute as people live longer. Results of our experiments demonstrate that oral 9-cis-retinoid is useful as a long-term prophylactic agent and as a therapeutic compound.
The beneficial effects and relative safety of 9-cis-retinoids extends to age-related macular degeneration, the leading cause of legal blindness in the U.S. and Europe (Zack, D. J., et al., Mol Vis 5, 30 (1999)). Here, we have shown that there are biochemical changes in the visual cycle that occur with age, namely an increase in free opsin and the opsin/rhodopsin ratio. When such biochemical changes are excessive they can lead to retinal degenerations such as seen in LCA (Fan, J., Woodruff, M. L., Cilluffo, M. C., Crouch, R. K. & Fain, G. L. J Physiol 568, 83-95 (2005)). In addition, free opsin results in spontaneous initiation of the visual cascade in rod photoreceptors (Lisman, J. & Fain, G. Nat Med 1, 1254-1255 (1995); Fain, G. L., Matthews, H. R., Cornwall, M. C. & Koutalos, Y. Physiol Rev 81, 117-151 (2001); Surya, A., Foster, K. W. & Knox, B. E. J Biol Chem 270, 5024-5031 (1995); Hofmann, K. P., Pulvermuller, A., Buczylko, J., Van Hooser, P. & Palczewski, K. J Biol Chem 267, 15701-15706 (1992); Palczewski, K., et al. Biochemistry 33, 13741-13750 (1994); Jager, S., Palczewski, K. & Hofmann, K. P. Biochemistry 35, 2901-2908 (1996)). This results in a decreased signal-to-noise ratio in the visual system, increased metabolic overload in the RPE, resulting in the formation of more waste products, free radicals and, eventually, drusen. Decreasing free opsin with 9-cis-retinoid therapy will therefore lead to a reduction in the precursors that are believed to initiate AMD.
Methods
Animals
Pigmented age-matched C57BL/6 female mice obtained from Charles River Laboratories were maintained on a normal diet in complete darkness or on a 12-hr light/dark cycle. All animal experiments utilized procedures approved by the University of Washington and Case Western Reserve University Animal Care Committees and conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia and the Association of Research for Vision and Ophthalmology.
9-cis-R-Ac was prepared as previously described (Batten, M. L., et al. PLoS medicine 2, e333 (2005); Batten, M. L., et al. J Biol Chem 279, 10422-10432 (2004)). A dose of ˜80 mg/kg body weight of 9-cis-R-Ac in 150 μl vegetable oil was administered via gavage to each treated animal. Prior to single dose experiments (
For the long-term study (
Two weeks after the last gavage with either, 9-cis-R-Ac, all-trans-R-Ac or vehicle, groups of 48 hr dark-adapted mice were analyzed for recovery of dark adaptation after light exposure, purified rhodopsin/opsin and retinoids in the eye and retinal morphology (FIG. 1C; Methods). Mice were exposed to light of intensity 500 cd·m−2 that bleached ˜90% of rhodopsin, anesthetized and monitored by ERG for one hr to evaluate recovery of dark adaptation. Rhodopsin was purified and rhodopsin/opsin ratios was determined. Retinoid analyses were performed on dissected eyes removed at 0, 10, 30, and 60 min after exposure to the same amount of light. Mice used to evaluate retinal morphology were not exposed to photobleaching prior to analysis.
Electroretinography (ERG)
ERGs of anesthetized mice were recorded as previously reported.
Purification of Rhodopsin
Rhodopsin purification was done under dim red light as previously described (Zhu, L., et al. J Biot Chem, 279, 53828-53839 (2004)). Purified anti-rhodopsin C terminus antibody 1D4 (MacKenzie, D., Arendt, A., Hargrave, P., McDowell, J. H. & Molday, R. S. Biochemistry, 23, 6544-6549 (1984)) was immobilized on CNBr-activated Sepharose 4B and a 4.6×12-mm column was packed with 2 mg of 1 D4 antibody/ml of Sepharose beads. Mouse whole eyes were homogenized in 137 mM NaCl, 5.4 mM Na2HPO4, 2.7 mM KCl and 1.8 mM KH2PO4 (pH 7.5) with a glass-to-glass homogenizer. Soluble proteins in the supernatant were removed by centrifugation at 14,000×g for 5 min and the pellet was solubilized in buffer containing 1% dodecyl-β-maltoside in 10 mM Bis-Tris propane (pH 7.5) containing 500 mM NaCl. The supernatant was cleared by centrifugation at 125,000×g for 20 min and loaded onto an antibody 1 D4-packed immunoaffinity column which was then thoroughly washed at a flow rate of 0.5 ml/min with 10 mM Bis-Tris propane (pH 7.5) containing 500 mM NaCl and 0.1% dodecyl-β-maltoside. Purified mouse rhodopsin was eluted with 100 μM nonapeptide (TETSQVAPA) in 10 mM Bis-Tris propane (pH 7.5) containing 500 mM NaCl and 0.1% dodecyl-β-maltoside at room temperature. Purified rhodopsin concentration was determined at 500 nm and total amount of opsin and rhodopsin at 280 nm with a Hewlett-Packard 8452A UV-visible spectrophotometer (Palczewski, K., Carruth, M. E., Adamus, G., McDowell, J. H. & Hargrave, P. A. Vision Research, 30, 1129-1137 (1990)).
Analysis of Retinoids
All experimental procedures related to extraction, derivatization and separation of retinoids were carried out under dim red light provided by a Kodak No. 1 safelight filter (transmittance>560 nm) as described previously (Van Hooser, J. P., et al. Proceedings of the National Academy of Sciences of the United States of America 97, 8623-8628 (2000); Van Hooser, J. P., et al. J Biol Chem, 277, 19173-19182 (2002); Maeda, A., et al. J Biol Chem 280, 18822-18832 (2005); Van Hooser, J. P., Garwin, G. G. & Saari, J. C. Methods Enzymol 316, 565-575 (2000)). Eluted fractions of purified rhodopsin from 6 mouse eyes were combined (total 3.0 ml) and mixed with an equal volume of 100% methanol. The mixture was vortexed and incubated on ice for 15 min. Retinoids were extracted twice with an equal volume of 100% hexane (6 ml total). The combined extracts were dried under argon and retinoids were separated by normal phase HPLC (Beckman, Ultrasphere-Si, 5 μm, 4.6×250 mm) with 10% ethyl acetate and 90% hexane at a flow rate of 1.4 ml/min and detected at 325 nm by an HPI 100 HPLC with a diode array detector and HP Chemstation A.03.03 software. A2E was analyzed as previously described (Maeda, A., et al. J Biol Chem, 280, 18822-18832 (2005)).
Analysis of retinoic acid in the liver was carried out as described before (Batten, M. L., et al. PLoS Medicine 2, e333 (2005)) by an Agilent 1100 HPLC with two tandem normal phase columns: a Varian Microsorb Silica 3 μm, 4.6×100 mm (Varian, Palo Alto, Calif.) and an Ultrasphere-Si, 5 μm, 4.6×250 mm column. An isocratic solvent system of 1000:4.3:0.675 hexane:2-propanol:glacial acetic acid (v/v) was used at a flow rate of 1 ml/min at 20° C. with detection at 355 nm. Calibration was done with standards of all-trans-RA and 9-cis-RA purchased from Sigma-Aldrich.
Immunoblotting
Immunoblotting was done according to standard protocols using Immobilon-P to adsorb proteins (polyvinylidene difluoride; Millipore Corp.). Monoclonal anti-rhodopsin antibody (1D4) was provided by Dr. R. Molday. The anti-LRAT (mAb) (Moise, A. R., Golczak, M., Imanishi, Y. & Palczewski, K., J Biol Chem (2006)), anti-transducin (Gt) (mAb) (unpublished), anti-guanylate cyclase 1 (1S4, mAb); Haire, S. E., et al. Investigative ophthalmology & visual science 47, 3745-3753 (2006)), anti-guanylate cyclase-activating protein 1 (UW14 pAb) (Gorczyca, W. A., et al. J Biol Chem, 270, 22029-22036 (1995)), anti-guanylate cyclase-activating protein 2 (UW50 pAb) Otto-Bruc, A., et al. Proceedings of the National Academy of Sciences of the United States of America 94, 4727-4732 (1997)), anti-rhodopsin kinase (Zhao, X., Huang, J., Khani, S. C. & Palczewski, K. J Biol Chem 273, 5124-5131 (1998)), and anti-retinol dehydrogenase 12 (pAb) (Maeda, A., et al. J Biol Chem 281, 37697-37704 (2006)) were generated in our laboratory. Alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Promega) were used as secondary antibodies. Protein bands were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium color development substrate (Promega). Proteins (30 μg per each well) were separated by 12.5% SDS-PAGE.
Retinal Morphology
For light microscopy, mouse eyecups were fixed with 2.5% glutaraldehyde and 1.6% paraformaldehyde in 0.08 M 1,4-piperazinediethanesulfonate buffer (PIPES) (pH 7.4) containing 2% sucrose for ˜1 hr at room temperature followed by 23 hr at 4° C. Eyecups then were washed with 0.13 M sodium phosphate buffer (pH 7.3) and dehydrated through a CH3OH series and embedded in JB4 glycol metacrylate. Sections (6 μm) were stained by immersion in 5% Richardson's stain for 1.5-2 min at room temperature and destained in 0.13 M sodium phosphate (pH 7.3) until the retinal layers were visible by light microscopy (about 8-15 min). For transmission electron microscopy, mouse eyecups were analyzed as described previously (Maeda, A., et al. J Blot Chem 280, 18822-18832 (2005); Maeda, T., Lem, J., Palczewski, K. & Haeseleer, F. Investigative ophthalmology & visual science 46, 4320-4327 (2005)).
DNA Microarray Analysis
RNA was isolated from 10 eyes, 100 mg of liver or 100 mg of kidney from groups C2 and N2 mice (
Array data for samples across the whole study were normalized by NimbleGen Systems Inc. (Madison, Wis.) that employed the robust multichip analysis feature of the data analysis package contained in the Bioconductor open source and open development software project for the analysis and comprehension of genomic data. Project-wide spreadsheets of robust multichip analysis results were exported to MICROSOFT® EXEL® and expression level ratios were calculated for all the possible pair-wise comparisons comprising one control and one treated sample. These pair-wise ratios were imported to Microsoft Access and mined for credible-fold changes in gene expression. Changes greater than or equal to a 2-fold increase or less than or equal to a 0.5-fold decrease were considered significant. Differentially expressed genes were then exported from Access as Excel files and were assigned functional annotations by LUCIDYX SEARCHER™ software by Lucidyx LLC.
Statistical Analyses
Statistical analyses were performed by one-way analysis of variance (ANOVA).
The previous examples were provided to illustrate but not to limit the scope of the claimed inventions. Other variants of the inventions will be readily apparent to those of ordinary skill in the art and are encompassed by the appended claims.
All publications, patents, patent applications and other references cited herein are hereby incorporated by reference, as allowed.
This application is a continuation of U.S. patent application Ser. No. 13/619,947, filed Sep. 14, 2012, now U.S. Pat. No. 9,408,821, which is a continuation of U.S. patent application Ser. No. 12/368,427, filed Feb. 10, 2009, now U.S. Pat. No. 8,324,270, which claims the benefit of priority to U.S. Provisional Patent Application No. 61/027,625, filed on Feb. 11, 2008, each of which is incorporated herein by reference in its entirety.
This invention was made with government support under grants EY008061 and EY013385 awarded by the National Institutes of Health. The government has certain rights in the invention.
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| 20160331713 A1 | Nov 2016 | US |
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| 61027625 | Feb 2008 | US |
| Number | Date | Country | |
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| Parent | 13619947 | Sep 2012 | US |
| Child | 15220315 | US | |
| Parent | 12368427 | Feb 2009 | US |
| Child | 13619947 | US |
