Patent application title: USE OF SERUM AMYLOID A GENE IN DIAGNOSIS AND TREATMENT OF GLAUCOMA AND IDENTIFICATION OF ANTI-GLAUCOMA AGENTS
Abbot F. Clark (Arlington, TX, US)
Wan-Heng Wang (Fort Worth, TX, US)
Wan-Heng Wang (Fort Worth, TX, US)
Loretta Graves Mcnatt (Hurst, TX, US)
Loretta Graves Mcnatt (Hurst, TX, US)
IPC8 Class: AA61K31437FI
Class name: Ring nitrogen in the additional hetero ring plural ring nitrogens in the additional hetero ring (e.g., imidazole, pyrazine, etc.) the ring nitrogens are bonded directly to each other (e.g., pyridazine, etc.)
Publication date: 2010-03-11
Patent application number: 20100063052
Patent application title: USE OF SERUM AMYLOID A GENE IN DIAGNOSIS AND TREATMENT OF GLAUCOMA AND IDENTIFICATION OF ANTI-GLAUCOMA AGENTS
Abbot F. Clark
Loretta Graves McNatt
Origin: FORT WORTH, TX US
IPC8 Class: AA61K31437FI
Patent application number: 20100063052
The present invention provides compositions and methods for treating
glaucoma, methods for diagnosing glaucoma, and methods for identifying
agents which may be useful in the treatment of glaucoma. More
specifically, the present invention describes the use of agents that
modulate the expression of serum amyloid A.
1. A method for treating glaucoma, said method comprising administering to
a patient in need thereof a therapeutically effective amount of a
composition comprising an agent that interacts with a gene encoding serum
amyloid A protein (SAA), wherein said interaction modulates the
expression of SAA, and wherein said agent inhibits p38 MAPkinase.
2. The method of claim 1, wherein the agent is selected from the group consisting of SB202190, SB203580, SB220025, PD 169316, SB 239063, 3-(-4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo-[3,2-b]pyridine, BIRB-796, and CalBio506126.
3. A method for treating glaucoma, said method comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that inhibits interaction of the serum amyloid A protein (SAA) with its receptor or modulates SAA downstream signaling events, wherein said agent inhibits p38 MAPkinase.
4. The method of claim 3, wherein said agent is selected from the group consisting of SB202190, SB203580, SB220025, PD 169316, SB 239063, 3-(-4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo-[3,2-b]pyridine, BIRB-796, and CalBio506126.
5. A pharmaceutical composition comprising a therapeutically effective amount of a serum amyloid A protein (SAA) antagonist and a pharmaceutical carrier, wherein the SAA antagonist is an inhibitor of p38 MAP kinase.
6. The composition of claim 5, wherein the p38 MAP kinase inhibitor is selected from the group consisting of SB202190, SB203580, SB220025, PD 169316, SB 239063, 3-(-4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo-[3,2-b]pyridine, BIRB-796, and CalBio506126.
The present invention is a continuation of U.S. patent application
Ser. No. 11/615,454, filed Dec. 22, 2006 (now allowed), which claims
priority from 11/000,757, filed Dec. 1, 2004 (now U.S. Pat. No.
7,357,931), which claims priority to U.S. provisional application No.
60/530,430, filed Dec. 17, 2003.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of diagnosis and treatment of glaucoma. More specifically, the invention provides methods and compositions for diagnosing and treating glaucoma and for identifying agents potentially useful for the treatment of is glaucoma.
2. Description of the Related Art
There are a number of ocular conditions that are caused by, or aggravated by, damage to the optic nerve head, degeneration of ocular tissues, and/or elevated intraocular pressure. For example, "glaucomas" are a group of debilitating eye diseases that are a leading cause of irreversible blindness in the United States and other developed nations. Primary Open Angle Glaucoma ("POAG") is the most common form of glaucoma. The disease is characterized by the degeneration of the trabecular meshwork, leading to obstruction of the normal ability of aqueous humor to leave the eye without closure of the space (e.g., the "angle") between the iris and cornea (Vaughan, D. et al., (1992)). A characteristic of such obstruction in this disease is an increased intraocular pressure ("IOP"), resulting in progressive visual loss and blindness if not treated appropriately and in a timely fashion. The disease is estimated to affect between 0.4% and 3.3% of all adults over 40 years old (Leske, M. C. et al. (1986); Bengtsson, B. (1989); Strong, N. P. (1992)). Moreover, the prevalence of the disease rises with age to over 6% of those 75 years or older (Strong, N. P., (1992)).
Glaucoma affects three separate tissues in the eye. The elevated IOP associated with POAG is due to morphological and biochemical changes in the trabecular meshwork (TM), a tissue located at the angle between the cornea and iris. Most of the nutritive aqueous humor exits the anterior segment of the eye through the TM. The progressive loss of TM cells and the build-up of extracellular debris in the TM of glaucomatous eyes leads to increased resistance to aqueous outflow, thereby raising IOP. Elevated IOP, as well as other factors such as ischemia, cause degenerative changes in the optic nerve head (ONH) leading to progressive "cupping" of the ONH and loss of retinal ganglion cells and axons. The detailed molecular mechanisms responsible for glaucomatous damage to the TM, ONH, and the retinal ganglion cells are unknown.
Twenty years ago, the interplay of ocular hypertension, ischemia and mechanical distortion of the optic nerve head were heavily debated as the major factors causing progression of visual field loss in glaucoma. Since then, other factors including excitotoxicity, nitric oxide, absence of vital neurotrophic factors, abnormal glial/neuronal interplay and genetics have been implicated in the degenerative disease process. The consideration of molecular genetics deserves some discussion insofar as it may ultimately define the mechanism of cell death, and provide for discrimination of the various forms of glaucoma. Within the past 10 years, over 15 different glaucoma genes have been mapped and 7 glaucoma genes identified. This includes six mapped genes (GLC1A-GLC1F) and two identified genes (MYOC and OPTN) for primary open angle glaucoma, two mapped genes (GLC3A-GLC3B) and one identified gene for congenital glaucoma (CYP1B1), two mapped genes for pigmentary dispersion/pigmentary glaucoma, and a number of genes for developmental or syndromic forms of glaucoma (FOXC1, PITX2, LMX1B, PAX6).
Thus, each form of glaucoma may have a unique pathology and accordingly a different therapeutic approach to the management of the disease may be required. For example, a drug that effects the expression of enzymes that degrade the extracellular matrix of the optic nerve head would not likely prevent RGC death caused by excitotoxicity. In glaucoma, RGC death occurs by a process called apoptosis (programmed cell death). It has been speculated that different types of insults that can cause death may do so by converging on a few common pathways. Targeting downstream at a common pathway is a strategy that may broaden the utility of a drug and increase the probability that it may have utility in the management of different forms of the disease. However, drugs that effect multiple metabolic pathways are more likely to produce undesirable side-effects. With the advent of gene-based diagnostic kits to identify specific forms of glaucoma, selective neuroprotective agents can be tested with the aim of reducing the degree of variation about the measured response.
Glaucoma is currently diagnosed based on specific signs of the disease (characteristic optic nerve head changes and visual field loss). However, over half of the population with glaucoma are unaware they have this blinding disease and by the time they are diagnosed, they already have irreversibly lost approximately 30-50% of their retinal ganglion cells. Thus, improved methods for early diagnosis of glaucoma are needed.
Current glaucoma therapy is directed to lowering IOP, a major risk factor for the development and progression of glaucoma. However, none of the current IOP lowering therapies actually intervenes in the glaucomatous disease process responsible for elevated IOP and progressive damage to the anterior segment continues. This is one possible reason why most patients become "resistant" to conventional glaucoma therapies. Thus, what is needed is a therapeutic method for altering (by inhibiting or even reversing) the disease process.
SUMMARY OF THE INVENTION
The present invention overcomes these and other drawbacks of the prior art by providing methods to diagnose and compositions to treat glaucoma. In one aspect, the present invention provides a method for treating glaucoma by administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that interacts with a gene encoding serum amyloid A protein (SAA), or with the gene's promoter sequence. The interaction between the agent and the gene encoding SAA, or with its promoter sequence, modulates the expression of SAA, such that the patient's glaucomatous condition is treated. In preferred embodiments, the agent will be a protein, peptide, peptidomimetic, small molecule or nucleic acid.
In another aspect, the present invention provides a method for treating glaucoma by administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that inhibits interaction of the serum amyloid A protein (SAA) with its receptor. Preferably, the agent will be a peroxisome proliferator-activated receptor α (PPARα) agonists, tachykinin peptides and their non-peptide analogs or α-lipoic acid. Most preferably, the agent will be fenofibrate, Wy-14643, (4-chloro-6-(2,3-xylidino)-2-pryrimidinylthiol)-acetic acid), ciprofibrate, 2-bromohexadecanoic acid, bezafibrate and ciglitizone, bafilomycin, concanamycin or pseudolaric acid B.
The present invention further provides a pharmaceutical composition for treating glaucoma comprising a therapeutically effective amount of a serum amyloid A protein (SAA) antagonist and a pharmaceutical carrier. The antagonist contained in the composition may be any of the compounds identified above.
In yet another embodiment, the present invention provides a method for diagnosing glaucoma, by the following steps: a) obtaining a biological sample from a patient; and b) analyzing said sample for an aberrant level, aberrant bioactivity or mutations of the gene encoding serum amyloid A protein (SAA) or its promoter region or its gene products, wherein said gene encoding SAA comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, wherein its promoter region comprises the sequence set forth in SEQ ID NO:12 or SEQ ID NO:13, and wherein SAA comprises the sequence set forth in SEQ ID NO:2 or SEQ ID NO:4; wherein the aberrantly high level, aberrantly high bioactivity or mutations of the SAA genes or the gene products indicates a diagnosis of glaucoma.
In preferred aspects, the biological sample is ocular tissue, tears, aqueous humor, cerebrospinal fluid, nasal or cheek swab or serum. Most preferably, the biological sample comprises trabecular meshwork cells.
Alternatively, the present invention provides a method for diagnosing glaucoma in a patient, by the steps: a) collecting cells from a patient; b) isolating nucleic acid from the cells; c) contacting the sample with one or more primers which specifically hybridize 5' and 3' to at least one allele of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:13 under conditions such that hybridization and amplification of the allele occurs; and d) detecting the amplification product; wherein aberrant level or mutations of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:13, in the sample indicates a diagnosis of glaucoma.
The present invention also provides a method for identifying agents potentially useful for treating glaucoma, by the steps: a) obtaining cells expressing SAA (SEQ ID NO:1 or SEQ ID NO:2) or cells containing SAA promoter/reporter gene such that the reporter gene is expressed; b) admixing a candidate substance with the cells; and c) determining the level of SAA protein (SEQ ID NO:2 or SEQ ID NO:4) or the level of gene expression in the cells; wherein an increase or decrease of the production of SAA protein or gene expression in the presence of said candidate substance indicates an agent potentially useful for the treatment of glaucoma.
In another aspect, the present invention provides a method for identifying an agent potentially useful for treating glaucoma, by the steps: a) forming a reaction mixture comprising: (i) an SAA protein or a cell expressing SAA or a reporter gene driven by an SAA promoter; (ii) an SAA protein binding partner; and (iii) a test compound; and b) detecting interaction of the SAA protein and binding partner or level of reporter gene products in the presence of the test compound and in the absence of the test compound;
wherein a decrease or increase in the interaction of the SAA protein with its binding partner in the presence of the test compound relative to the interaction in the absence of the test compound indicates a potentially useful agent for treating glaucoma.
In another aspect, the present invention provides a method for identifying an agent potentially useful for treating glaucoma, by the steps: a) forming a reaction mixture comprising: (i) cells comprising SAA recombinant protein (SEQ ID NO:2 or SEQ ID NO:4) or cells comprising expression vectors comprising SEQ ID NO:1 or SEQ ID NO:3; and (ii) a test compound; and b) detecting the effect on downstream signalling (IL-8) in the presence of the test compound and in the absence of the test compound;wherein a decrease or increase in the downstream signalling in the presence of the test compound relative to the interaction in the absense of the test compound indicates a potentially useful agent for treating glaucoma.
In preferred aspects, the cells containing the SAA protein or expression vectors will be HL-60 cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. QPCR analysis of SAA expression in 12 glaucoma vs. 11 normal TM tissues. NTM and GTM represent average expression level of the gene in normal and glaucoma groups, respectively.
1© FIG. 2A. QPCR analysis of SAA expression in TM cell lines. NTM and GTM represent average expression level of the gene in normal and glaucoma groups, respectively.
FIG. 2B. QPCR analysis of SAA expression in optic nerve head tissues. NTM and GTM represent average expression level of the gene in normal and glaucoma groups, respectively.
FIG. 3. SAA protein in TM tissues from normal and glaucoma donors (n=6). A significant increase (3-fold) in SAA was observed in glaucoma TM tissues compared to normal tissue (p=0.05). The bars show mean+/-s.e.m.
FIG. 4. SAA protein determined by ELISA in human aqueous humor from normal and glaucomatous individuals. The values are expressed as the average SAA in ng/ml of aqueous humor, +/-s.e.m. (p=0.0001).
FIG. 5. IL-8 secretion by HL-60 cells in response to increasing concentrations of rhSAA.
FIG. 6. Effect of Adv.SAA2 on mouse IOP by intravitreal injection. IOP was measured with the rebound tonometers TonoLab®.
FIG. 7. SAA expression in mouse eyes from Balb/c mice 28 days after intravitreal injection. SAA was measured by ELISA. Control: Adv.null injected eyes and no injection eyes (contralateral eyes of Adv.SAA2 injected eyes; n=16); Adv.SAA: Adv.SAA2 injected eyes (n=17).
FIG. 8A and FIG. 8B. Effect of intravitreal injection of Ad.SAA2+anti-CD40L antibody on Balb/c mouse IOP (FIG. 8A) and iris hyperemia (FIG. 8B). Data are presented as mean and SEM.
FIG. 9. Effect of recombinant human Serum Amyloid A (rhSAA, 1 μg/mL; treatment started at time 0) on IOP of perfused human anterior segments.
FIG. 10. Effect of recombinant human Serum Amyloid A (rhSAA, 1 μg/mL; treatment started at time 0) on interleukin-8 (IL-8) level in the perfusate of perfused human anterior segments.
FIG. 11. Effect of MAP p38 kinase inhibitors on induction of IL-8 by SAA treatment (1 μg/ml) in TM cells. A: Effect of SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole- ). B: Effect of 4-azaindole and BIRB-796 (1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)- naph-thalen-1-yl)urea) at 50 μM. IL-8 was measured in the media by ELISA.
FIG. 12. Inhibition of SAA stimulated IL-8 secretion by SB203580 in TM cells and HL-60 cells. NTM650-03, p9 or HL-60 cells were treated in serum-free DMEM for 4 hours with 1 μg/ml SAA and the indicated concentrations of SB202580. IL-8 was measured in the media by ELISA. Calculated IC50=15 μM in TM cells and 25 μM in HL-60 cells.
FIG. 13. Inhibition of SAA stimulated IL-8 secretion by the p38 MAP kinase inhibitors, 4-azaindole and BIRB-796 in HL60 cells treated in serum-free DMEM for 4 hours with 1 μg/ml SAA and the indicated concentrations of inhibitors. IL-8 was measured in the media by ELISA. Calculated IC50: 4-azaindole=0.3 μM, BIRB-796=0.5 μM.
DETAILED DESCRIPTION PREFERRED EMBODIMENTS
Glaucoma is a heterogeneous group of optic neuropathies that share certain clinical features. The loss of vision in glaucoma is due to the selective death of retinal ganglion cells in the neural retina that is clinically diagnosed by characteristic changes in the visual field, nerve fiber layer defects, and a progressive cupping of the ONH. One of the main risk factors for the development of glaucoma is the presence of ocular hypertension (elevated intraocular pressure, IOP). IOP also appears to be involved in the pathogenesis of normal tension glaucoma where patients have what is often considered to be normal IOP. The elevated IOP associated with glaucoma is due to elevated aqueous humor outflow resistance in the trabecular meshwork (TM), a small specialized tissue located in the iris-corneal angle of the ocular anterior chamber. Glaucomatous changes to the TM include a loss in TM cells and the deposition and accumulation of extracellular debris including proteinaceous plaque-like material. In addition, there are also changes that occur in the glaucomatous optic nerve head (ONH). In glaucomatous eyes, there are morphological and mobility changes in ONH glial cells. In response to elevated IOP and/or transient ischemic insults, there is a change in the composition of the ONH extracellular matrix and alterations in the glial cell and retinal ganglion cell axon morphologies.
The present inventors have discovered that the expression of Serum Amyloid A (SAA) mRNA and protein are significantly upregulated in glaucomatous TM tissues and cells. The inventors have verified the differential mRNA expression seen using Affymetrix gene chips by real time quantitative polymerase chain reaction (QPCR) and increased SAA protein levels by SAA ELISA. This is the first time SAA has been shown to be expressed in the TM.
Human SAA comprises a number of small, differentially expressed apolipoproteins encoded by genes localized on the short arm of chromosome 11. There are four isoforms of SAAs. SAM (SEQ ID NO:2), encoded by SEQ ID NO:1, and SAA2 (SEQ ID NO:4), encoded by SEQ ID NO:3, are known as acute phase reactants, like C-reactive protein, that is, they are dramatically upregulated by proinflammatory cytokines. The 5'UTR promoter regions of SAM and SAA2 genes are also provided (SEQ ID NO:12 and SEQ ID NO:13, respectively). SAA3 (SEQ ID NO:5) is a pseudogene and SAA4 (SEQ ID NO:6) is a low level constitutively expressed gene encoding constitutive SAA4 (SEQ ID NO:7). SAA2 has two isoforms, SAA2α (SEQ ID NO:9), encoded by SEQ ID NO:8, and SAA2β (SEQ ID NO:11), encoded by SEQ ID NO:10, which differ by only one amino acid. SAA1 and SAA2 proteins are 93.5% identical at the amino acid level (SEQ ID NO:2 and SEQ ID NO:4, respectively) and these genes are 96.7% identical at the nucleotide level (SEQ ID NO:1 and SEQ ID NO:3, respectively).
SAA is an acute-phase reactant whose level in the blood is elevated approximately 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, extrahepatic SAA expression was described initially in mouse tissues, and later in cells of human atherosclerotic lesions (O'Hara et al. 2000). Subsequently, SAA mRNA was found widely expressed in many histologically normal human tissues. Localized expression was noted in a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. SAA protein expression co-localized with SAA mRNA expression has also been reported in histologically normal human extrahepatic tissues. (Liang et al. 1997; Urieli-Shoval et al. 1998).
SAA isoforms are apolipoproteins that become a major component of high-density lipoprotein (HDL) in the blood plasma of mammals and displaces A-I (ApoA-I) and phospholipid from the HDL particles (Miida et al. 1999). SAA binds cholesterol and may serve as a transient cholesterol-binding protein. In addition, over-expression of SAA1 or SAA2 leads to the formation of linear fibrils in amyloid deposits, which can lead to pathogenesis (Uhlar and Whitehead 1999; Liang et al. 1997). SAA plays an important role in infections, inflammation, and in the stimulation of tissue repair. SAA concentration may increase up to 1000-fold following inflammation, infection, necrosis, and decline rapidly following recovery. Thus, serum SAA concentration is considered to be a useful marker with which to monitor inflammatory disease activity. Hepatic biosynthesis of SAA is up-regulated by pro-inflammatory cytokines, leading to an acute phase response. Chronically elevated SAA concentrations are a prerequisite for the pathogenesis of secondary amyloidosis, a progressive and sometimes fatal disease characterized by the deposition in major organs of insoluble plaques composed principally of proteolytically cleaved SAA. This same process also may lead to atherosclerosis. There is a requirement for both positive and negative SAA control mechanisms to maintain homeostasis. These mechanisms permit the rapid induction of SAA expression to fulfill host-protective functions, but they also must ensure that SAA expression is rapidly returned to baseline levels to prevent amyloidosis. These mechanisms include modulation of promoter activity involving, for example, the inducer nuclear factor kB (NF-kB) and its inhibitor IkB, up-regulation of transcription factors of the nuclear factor for interleukin-6 (NF-IL6) family, and transcriptional repressors such as yin and yang 1 (YY1). Post-transcriptional modulation involving changes in mRNA stability and translation efficiency permit further up- and down-regulatory control of SAA protein synthesis to be achieved. In the later stages of the AP response, SAA expression is effectively down-regulated via the increased production of cytokine antagonists such as the interleukin-1 receptor antagonist (IL-1Ra) and of soluble cytokine receptors, resulting in less signal transduction driven by pro-inflammatory cytokines (Jensen and Whitehead 1998).
There are several reports suggesting that primary amyloidosis may be associated with glaucoma. For example, it was found that amyloid was deposited in various ocular tissues including the vitreous, retina, choroid, iris, lens, and TM in primary systemic amyloidosis patients (Schwartz et al. 1982). Ermilov et al. (1993) reported that in 478 eyes of 313 patients, aged 25 years to 90 years, with cataracts, glaucoma, and/or diabetes mellitus, 66 (14%) of the eyes contained amyloid-pseudoexfoliative amyloid (PEA). Krasnov et al. (1996) reported that 44.4% of 115 patients with open-angle glaucoma revealed extracellular depositions of amyloid. Amyloidosis was revealed in the sclera in 82% of the cases and in the iris in 70% of the cases. A number of clinical conditions, including Alzheimer's disease, exhibit aberrant amyloid tissue deposits associated with disease. However, amyloids are molecularly heterogeneous and encoded by different amyloid genes. The previous reports are unclear regarding which amyloid(s) might be associated with glaucoma. The present inventors have shown, for the first time, that SAA gene expression is elevated significantly in glaucomatous TM tissues. Increased SAA may be involved in the generation of elevated IOP and damage to the optic nerve leading to vision loss in glaucoma patients. The present invention provides methods of using a finding of increased SAA expression to diagnose glaucoma. The present invention further provides methods for screening for agents that alter SAA expression or function in order to identify potentially anti-glaucomatous agents. In another aspect, the present invention provides methods and compositions of using agents that antagonize SAA actions and/or interactions with other proteins for the treatment of glaucoma.
Based on the inventors' finding that certain subjects with glaucoma have increased levels of SAA expression, the present invention provides a variety of methods for diagnosing glaucoma. Certain methods of the invention can detect mutations in nucleic acid sequences that result in inappropriately high levels of SAA protein. These diagnostics can be developed based on the known nucleic acid sequence of human SAA, or the encoded amino acid sequence (see Miller 2001). Other methods can be developed based on the genomic sequence of human SAA or of the sequence of genes that regulate expression of SAA. Still other methods can be developed based upon a change in the level of SAA gene expression at the mRNA level.
In alternative embodiments, the methods of the invention can detect the activity or level of SAA signaling proteins or genes encoding SAA signaling proteins. For example, methods can be developed that detect inappropriately low SAA signaling activity, including for example, mutations that result in inappropriate functioning of SAA signaling components, including SAA induction of IL-8. In addition, non-nucleic acid based techniques may be used to detect alteration in the amount or specific activity of any of these SAA signaling proteins.
A variety of means are currently available to the skilled artisan for detecting aberrant levels or activities of genes and gene products. These methods are well known by and have become routine for the skilled artisan. For example, many methods are available for detecting specific alleles at human polymorphic loci. The preferred method for detecting a specific polymorphic allele will depend, in part, upon the molecular nature of the polymorphism. The various allelic forms of the polymorphic locus may differ by a single base-pair of the DNA. Such single nucleotide polymorphisms (or SNPs) are major contributors to genetic variation, comprising some 80% of all known polymorphisms, and their density in the human genome is estimated to be on average 1 per 1,000 base pairs. A variety of methods are available for detecting the presence of a particular single nucleotide polymorphic allele in an individual. Advancements in the field have provided accurate, easy, and inexpensive large-scale SNP genotyping. For example, see U.S. Pat. No. 4,656,127; French Patent 2,650,840; PCT App. No. WO91/02087; PCT App. No. WO92/15712; Komher et al. 1989; Sokolov 1990; Syvanen et al. 1990; Kuppuswamy et al. 1991; Prezant et al. 1992; Ugozzoli et al. 1992; Nyren et al. 1993; Roest et al. 1993; and van der Luijt et al. 1994).
Any cell type or tissue may be utilized to obtain nucleic acid samples for use in the diagnostics described herein. In a preferred embodiment, the DNA sample is obtained from a bodily fluid, e.g., blood, obtained by known techniques (e.g. venipuncture), or buccal cells. Most preferably, the samples for use in the methods of the present invention will be obtained from blood or buccal cells. Alternately, nucleic acid tests can be performed on dry samples (e.g. hair or skin).
Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo 1992).
In addition to methods which focus primarily on the detection of one nucleic acid sequence, profiles may also be assessed in such detection schemes. Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
A preferred detection method is allele specific hybridization using probes overlapping a region of at least one allele of an SAA signaling component that is indicative of glaucoma and having about 5, 10, 20, 25 or 30 contiguous nucleotides around the mutation or polymorphic region. In a preferred embodiment of the invention, several probes capable of hybridizing specifically to other allelic variants involved in glaucoma are attached to a solid phase support, e.g., a "chip" (which can hold up to about 250,000 oligonucleotides). Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe arrays" is described e.g., in Cronin et al. (1996). In one embodiment, a chip comprises all the allelic variants of at least one polymorphic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridisation to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.
These techniques may further include the step of amplifying the nucleic acid before analysis. Amplification techniques are known to those of skill in the art and include, but are not limited to, cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli et al. 1990), transcriptional amplification system (Kwoh et al. 1989), and Q-Beta Replicase (Lizardi, et al. 1988).
Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele-specific oligonucleotide (ASO) hybridization, allele specific 5' exonuclease detection, sequencing, hybridization, SSCP, and the like.
PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and thus can each be differentially detected. Of course, hybridization based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analyses of a plurality of markers.
In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize 5' and 3' to at least one allele of SAA that is indicative of glaucoma under conditions such that hybridization and amplification of the allele occurs, and (iv) detecting the amplification product. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In a preferred embodiment of the subject assay, aberrant levels or activities of SAA that are indicative of glaucoma are identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the allele. Exemplary sequencing reactions include those based on techniques developed my Maxim and Gilbert (1977) or Sanger (1977). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays, including sequencing by mass spectrometry (see, for example WO94/16101; Cohen et al. 1996; Griffin et al. 1993). It will be evident to one of skill in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleic acid is detected, can be carried out.
In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamin or osmium tetraoxide and with piperidine) can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes (Myers et al. 1985b; Cotton et al. 1988; Saleeba et al. 1992). In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes). For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T and G/T mismatches (Hsu et al. 1994; U.S. Pat. No. 5,459,039).
In other embodiments, alterations in electrophoretic mobility will be used to identify aberrant levels or activities of SAA that are indicative of glaucoma. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. 1989; Cotton 1993; Hayashi 1992; Keen et al. 1991).
In yet another embodiment, the movement of alleles in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. 1985a). In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner 1987).
Examples of other techniques for detecting alleles include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. 1986; Saiki et al. 1989). Such allele specific oligonucleotide hybridization techniques may be used to test one mutation or polymorphic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymorphic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation or polymorphic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. 1989) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner 1993). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. 1992). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany 1991). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
In another embodiment, identification of an allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, E.g., in U.S. Pat. No. 4,998,617 and in Landegren et al. 1988). Nickerson et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson et al. 1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Several techniques based on this OLA method have been developed and can be used to detect aberrant levels or activities of SAA that are indicative of glaucoma. For example, U.S. Pat. No. 5,593,826 and To be et al. (1996), describe such techniques that are frequently used.
In one embodiment, fenofibrate, a peroxisome proliferator-activated receptor a (PPARα) agonist, may be formulated in a pharmaceutically acceptable composition and used to treat glaucoma by modulating SAA expression. Studies have shown that fenofibrate and WY 14643 treatment reduces plasma SAA concentration (Yamazaki et al. 2002). It is believed that other PPARα agonists, such as ciprofibrate, 2-bromohexadecanoic acid, bezafibrate, ciprofibrate and ciglitizone may also be useful for the treatment of glaucoma.
In another embodiment, p38 MAP kinase inhibitors may be used to treat glaucoma by modulating SAA induced expression of IL-8 and downstream signaling events. The inventors showed that SAA stimulates secretion of IL-8 in trabecular meshwork cells and tissue. One pathway for upregulation of IL-8 is through activation of MAP kinases. The inventors further showed that inhibitors of p38 MAP kinase block SAA induction of IL-8 in TM cells, in perfusion cultured human eyes, and in vivo in rodent eyes. Compounds representing different classes of MAPK inhibitors in TM cells were found to be effective inhibitors of SAA induced IL-8 expression (Table 3). The most potent of these were the p38 MAPK inhibitors, SB203580, 4-azaindole, and BIRB-796 (FIG. 11). Dose response curves for SB203580 inhibition of SAA induced IL-8 generated in both TM cells and HL60 cells gave similar results (IC50=15 μM in TM cells and 25 μM in HL60 cells, FIG. 12). Inhibition curves for 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b]pyridine (also referred to herein as a 4-azaindole) and BIRB0796 conducted in HL60 cells showed both of these compounds to be approximately 10 times more effective than SB203580 (IC50=0.3 for 4-azaindole and 0.5 μM for BIRB-796 (FIG. 13).
TABLE-US-00001 TABLE 3 Compound Classes Screened for Inhibition of SAA Induced IL-8 Secretion in TM Cells % Inhibition of SAA induced IL-8 secretion Compound Target Selectivity 100 μM 50 μM 25 μM 10 μM 1 μM SB203580 MAPK p38 MAP kinase 98.7 73.8 61.0 38.1 16.3 SB202190 MAPK p38 MAP kinase 38.2 43.3 17.2 BIRB-796 MAPK p38 MAP kinase 78.1 28.9 4-azaindole MAPK p38 MAP kinase 94.3 22.6 PD98059 MAPK MEK 34.0 U0126 MAPK MEK 53.0 50.6 30.9 Fenofibrate SAA PPARα agonist 57.2 52.7 59.9 Chemical names for the compounds identified in Table 3 are as follows: SB203580: 4-(4-fluorophenyl)-2-(40methylsulfinylphenyl)-5-(4-pyridyl)-1H-i- midazole; SB202190: 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol; BIRB-796: 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl- -ethoxy)naph-thalen-1-yl)urea; 4-azaindole: 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine; PD98059: 2'-amino-3'-methoxyflavone; U0126: 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; CalBio506126: 2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3- -one
In vitro assays showed that several p38MAPK inhibitors significantly blocked IL-8 induction by SAA. 3-(4-fluorophenyl)-2-(pyridine-4-yl)-1H-pyrrolo-[3,2-b]pyridine, one of most potent p38 MAPK inhibitors was evaluated for lowering IOP in the mouse by topical application of a 1% suspension after intravitreal injection of Ad.SAA2 (2×107 pfu/eye)+antiCD40L. Adv.SAA2, caused a significant IOP elevation, which peaked at days 10-12 with 10-12 mmHg increase from baseline, followed by a slow decline. At day 24, the IOP was 6-7 mmHg above baseline. The ocular hypertension was blocked by topical administration of 4-azaindole (1%; b.i.d.). After 4-azaindole administration was stopped at Day 7, IOP returned to the same level as the vehicle-treated Ad.SAA2-injected group. When drug administration was resumed at day 13, IOP was again lowered to baseline in 3 days (FIG. 8). This experiment was repeated and similar results were obtained. The in vivo data showed that the p38MAPK inhibitor, 4-azaindole, can counteract ocular hypertension induced by Ad.SAA2.
Preferred compounds for this embodiment of the invention are those classes of is compounds listed in Table 3, and extends to additional classes of compounds that exibit inhibitory properties for p38MAP kinases as described in these examples and include SB202190, SB203580, SB220025, PD 169316, SB 239063, 4-azaindole, BIRB-796, CalBio506126.
The present inventors further postulate that agents that prevent amyloid-induced cell death may be useful for protecting TM and other ocular cells in the anterior uvea and at the back of the eye, especially the retina and optic nerve head.
The Compounds of this invention, can be incorporated into various types of ophthalmic formulations for delivery to the eye (e.g., topically, intracamerally, or via an implant). The Compounds are preferably incorporated into topical ophthalmic formulations for delivery to the eye. The Compounds may be combined with opthalmologically acceptable preservatives, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, and water to form an aqueous, sterile ophthalmic suspension or solution. Ophthalmic solution formulations may be prepared by dissolving a Compound in a physiologically acceptable isotonic aqueous buffer. Further, the ophthalmic solution may include an opthalmologically acceptable surfactant to assist in dissolving the Compound. Furthermore, the ophthalmic solution may contain an agent to increase viscosity, such as, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, or the like, to improve the retention of the formulation in the conjunctival sac. Gelling agents can also be used, including, but not limited to, gellan and xanthan gum. In order to prepare sterile ophthalmic ointment formulations, the active ingredient is combined with a preservative in an appropriate vehicle, such as, mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations may be prepared by suspending the Compound in a hydrophilic base prepared from the combination of, for example, carbopol-974, or the like, according to the published formulations for analogous ophthalmic preparations; preservatives and tonicity agents can be incorporated.
The Compounds are preferably formulated as topical ophthalmic suspensions or solutions, with a pH of about 4 to 8. The establishment of a specific dosage regimen for each individual is left to the discretion of the clinicians. The Compounds will normally be contained in these formulations in an amount 0.01% to 5% by weight, but preferably in an amount of 0.05% to 2% and most preferably in an amount 0.1 to 1.0% by weight. The dosage form may be a solution, suspension microemulsion. Thus, for topical presentation 1 to 2 drops of these formulations would be delivered to the surface of the eye 1 to 4 times per day according to the discretion of a skilled clinician.
The Compounds can also be used in combination with other agents for treating glaucoma, such as, but not limited to, β-blockers, prostaglandins, carbonic anhydrase inhibitors, α2 agonists, miotics, and neuroprotectants.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Increased Expression of Sam and SAA2 in Glaucomatous TM Cells and Tissues
RNA pools of TM tissues from 13 normal donors vs. 9 glaucoma donors was used to determine gene expression using the Affymetric GeneChips set (HG-U133). Amyloid A2 expression was identified to increase 4 fold in glaucoma comparing to that in normal TM tissues. To confirm this result, QPCR was conducted using individual RNA from 12 glaucoma and 11 normal TM tissues. Five from 12 glaucoma TM tissues (42%) showed significant increase in SAA1/2 expression. Average of SAA expression in the 12 glaucoma TM was 5.4 fold to that in the 11 normal TM (FIG. 1). In addition, a similar trend of SAA differential expression was observed in glaucoma TM cells or glaucoma optic nerve head tissues. There was an average increase of 5.4-fold in glaucoma TM cells (14 glaucoma vs. 11 normal TM cell lines, FIG. 2A) and 118-fold in glaucoma optic nerve head tissues (14 glaucoma vs. 12 normal, FIG. 2B) compared to normals, respectively. ELISA of SAA in TM tissues from 6 normal and 6 glaucoma donors showed that SAA protein was also significantly increased in glaucoma TM tissues compared to normals. There was a 3-fold difference in SAA concentration in glaucomatous tissue compared to normal tissue (11.3 and 3.8 μg/mg protein respectively). These data are shown in FIG. 3.
An association of increased expression of SAA with glaucoma was further demonstrated in human aqueous humor. SAA protein was measured by ELISA in aqueous humor from 16 normal and 20 glaucomatous individuals. SAA was found to be almost 3 times higher in glaucomatous aqueous humor than in normal samples (10.0 ng/ml vs. 3.7 ng/ml respectively). The results are shown in FIG. 4.
Formulation of Fenofibrate for Topical Application
1% Fenofibrate suspension for topical application to decrease SAA and lower IOP in the eye.
TABLE-US-00002 Description Conc. Units Purpose Fenofibrate, NOC 1% W/V % active ingredient hydroxypropyl methyl- 0.5% W/V % viscosity modifier cellulose (2910) (E4M), USP dibasic sodium phosphate 0.2% W/V % buffering agent (anhydrous), usp sodium chloride, usp 0.75% W/V % tonicity agent disodium edta 0.01% W/V % chelating agent (edetate disodium), usp polysorbate 80, nf 0.05% W/V % wetting agent benzalkonium chloride, nf 0.01% W/V % preservative sodium hydroxide, nf q.s. pH W/V % pH adjust hydrochloric acid, nf q.s. pH W/V % pH adjust purified water, usp q.s. 100% W/V % vehicle
Procedure for Screening and Identifying Compounds that Alter the Expression of SAA mRNA or SAA Proteins
One method that can be used for screening for agents that alter SAA expression and function is to determine changes in SAA protein levels. Kits for in vitro assay for quantitative determination of Serum Amyloid A (SAA) in animal or human sera, plasma, buffered solutions, cell culture media, and tissue or cell extracts are commercially available. The assay is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for SAA has been coated onto the wells of a microtiter plate. Samples, including standards of known SAA content, or unknowns, are added to these wells along with a secondary antibody conjugated to alkaline phosphatase or peroxidase. The antibodies are constructed such that neither one interferes with the binding epitope of the other. The SAA is both captured on the plate by the immobilized antibody and labeled with the conjugated second antibody in a one step procedure. After an incubation period, the plate is washed to remove all unbound material and a substrate (PNPP or peroxide) is added. The intensity of the colored product is proportional to the concentration of SAA present in the unknown sample.
Induction of SAA in Cultured Cell Lines for Screening Compounds that Alter the Expression of SAA mRNA or Protein
The human hepatoma cell line, HepG2, is widely used for studies on SAA induction by cytokines, for transfection with plasmids, and reporter assays. SAA mRNA and protein synthesis can be induced by various cytokines in several human hepatoma cell lines including PCL/PRF/5, HepB and HepG2 (Uhlar and Whitehead 1999). SAA synthesis by human aortic smooth muscle cells (HASMC) is induced by glucocorticoid hormones and not by the proinflammatory cytokines, IL-1, IL-6, and TNF-α, which stimulate the production of SAA by hepatocytes (Kumon et al. 2002b; Kumon et al. 2001; Thorn and Whitehead 2002). SAA stimulated the chemotactic migration of HASMC in a dose dependent manner when assayed using a Chemotaxicell culture chamber (Kumon et al. 2002a). SAA mRNA expression and protein production was demonstrated in primary cultures of rheumatoid arthritis synoviocytes (O'Hara et al. 2000).
Functional Analysis of SAA in Cultured Cells
Cytokine-like properties of SAA include induction of IL-8 secretion by neutrophils. (Furlaneto and Campa, 2002; He et al. 2003). HL-60 cells, a promyelocytic cell line, was identified that responds to SAA with increased IL-8 secretion, and can be used for in vitro assays of SAA function. HL-60 cells were treated for four hours with increasing concentrations of recombinant human SAA, and IL-8 was measured in the media by ELISA. IL-8 secretion increased in a dose dependent manner (FIG. 5). HL-60 cells can be used as a surrogate cell line for functional assays to identify agents that alter SAA function and expression levels.
Adenovirus Mediated SAA Expression Increases IOP and P38 MAPK Inhibitor Decreases the Induced IOP in the Mouse
The ability of upregulated SAA expression through adenovirus to elevate IOP and efficacy of p38 MAPK inhibitors in blockage of the induced IOP in mouse was studied.
1. Upregulated SAA Expression Elevates IOP in Mouse
One eye of each Balb/c mice was intravitreally injected with Adv.SAA2 (treatment) or Adv.null (vehicle) at dosage of 7×107 pfu/eye/2ul. Contralateral eye of each animal was not injected. In addition to Adv, each animal received IP injection of anti-CD40L (0.5 mg/injection) on days -1, 0, 1, 2, 5, 9, & 14 to prolong the expression period of the Adv.SAA2. Mouse IOP was measured by Tonolab in a mask way. Mean of IOP for each eye was obtained from 18 to 30 measurements. Intravitreal injection of Adv.SAA2 in mice significantly increased IOP (49% or 5.8 mm Hg, n=6-8, p<0.05) (FIG. 6). SAA expression was significantly higher in all Adv.SAA2 treated eyes than that in control eyes including vehicle treated eyes and contralateral eyes without injection (p<0.0001; n=16) (FIG. 7). These results demonstrated that upregulating SAA expression can increase mouse IOP, providing evidence of SAA linkage to glaucoma pathogenesis.
2. p38 MAPK Inhibitor Lowers Adv.SAA2 Induced IOP in Mouse:
After establishing that 4-azaindole, a P38 MAPK inhibitor inhibits the SAA-induced IL-8 expression in vitro, the inventors tested the effect of the compound on mouse IOP after intravitreal injection of Ad.SAA2 (2×107 pfu)+antiCD40L by topical administration of 5 μL of 1% 4-azaindole or vehicle on both eyes, b.i.d., from day -1 to day 7 and day 13 to day 17. Again, intravitreal injection of Adv.SAA2 significantly increased mouse IOP from days 4 to 24 (vehicle group). Topical dosing of 4-azaindole significantly inhibited the Adv.SAA2-induced IOP during the period of treatment time (days -1 to 7 and days 13 to 17. 4-azaindole did not affect IOP in the non-injected eyes (FIG. 8A). Iris hyperemia was observed in all Ad.SAA2-injected eyes after day 4 and slowly decreased during the second week after injection (FIG. 8B). 4-azaindole did not affect hyperemia in the injected eyes, indicating the IOP-lowing effect of 4-azaindole was not through dispelling of iris hyperemia. These results demonstrate the potential of p38 MAPK inhibitors for treatment of ocular hypertension.
Recombinant SAA Decreased Outflow Facility in Perfusion Cultured Human Eyes
Five pairs of human eyes were perfused with media containing either recombinant SAA (1 μg/mL) (experimental eye) or an equivalent volume of vehicle (control eye). At the end of the culture period, 4 quadrants of each eye were examined by transmission electron microscopy to determine TM tissue viability. Perfusate of each eye was collected and used for ELISA measurement of IL-8 level. All five had an elevated IOP within 24 h of treatment (FIG. 9). All five had an elevated IL-8 level (FIG. 10). The change in IOP correlated with SAA-induced increase in interleukin-8 in the perfusates. All five pairs of eyes had acceptable post-perfusion TM viability scores. These results demonstrated that increased SAA level can elevate IOP in perfusion cultured human eyes.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and structurally related may be substituted for the agents described herein to achieve similar results. All such substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Furlenato, C J, and Campa A, A novel function of serum amyloid A: a potent stimulus for the release of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 by human blood neutrophil, BIOCHEM. BIOPHYS. RES. COMMUN 268:405-408 (2002). He, R, Sang H, Ye, R D, Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R, BLOOD 101:1572-1581 (2003). Jensen L E and Whitehead A S, BIOCHEM. J. 334:489-503 (1998). Jordat M S, et al., PLANTA MED. 68:667-71 (2002). Kane et al., J. NEUROCHEM., 72: 1939-1947 (1999). Kumon, Y., Hosokawa, T., Suchiro, T., Ideda, Y., Sipe, J. D., and Hashimoto, K., Acute-phase, but not constitutive serum amyloid A (SAA) is chemotactic for cultured human aortic smooth muscle cells, AMYLOID 9:237-241 (2002a). Kumon, Y., Suchiro, T., Faulkes, D. J., Hosakawa, T., Ideda, Y., Woo, P., Sipe, J. D., and Hashimoto, K., Transcriptional regulation of Serum Amyloid A1 gene expression in human aortic smooth muscle cells involves CCAAT/enhancer binding proteins (C/EBP) and is distinct from HepG2 cells, SCAND. J. IMMUNOL. 56:504-511 (2002b). Kumon, Y., Suchiro, T., Hashimoto, K., and Sipe, J. D., Dexamethasone, but not IL-1 alone, upregulates acute-phase serum amyloid A gene expression and production by cultured human aortic smooth muscle cells, SCAND J. IMMUNOL. 53:7-12 (2001). Lambert et al., PROC. NAT. ACAD. SCI. USA 95: 6448-6453 (1998). Liang, J. S., Sloane, J. A., Wells, J. M., Abraham, C. R., Fine, R. E., and Sipe, J. D., Evidence for local production of acute phase response apolipoprotein serum amyloid A in Alzheimer's disease brain, NEUROSCI. LETT. 225:73-76 (1997). Liu et al., J. NEUROCHEM. 69: 2285-2293 (1997). Miida T., Yamada, T., Yamadera, T., Ozaki, K., Inano, K., Okada, M., Serum amyloid A protein generates pre-beta 1 high-density lipoprotein from alpha-migrating high-density lipoprotein, BIOCHEM. 38(51):16958-16962 (1999). Miller, Genome Biology 3 (1):reviews 3001.1-3001.15 (2001) (also at http://genomebiology.com/2001/3/1/reviews/3001.1) Nakagami et al., EUR. J. PHARMACOL. 457: 11-17 (2002a). Nakagami et al., BR. J. PHARMACOL., 137: 676-682 (2002b). O'Hara, R., Murphy, E. P., Whitehead, A. S., FitzGerald, O., and Bresnihan, B., Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue, ARTHRITIS RES. 2:142-144 (2000). Pike et al., J. NEUROSCI. 13: 1676-1687 (1993). Thorn, C. F. and Whitehead, A. S., Differential glucocorticoid enhancement of the cytokine-driven transcriptional activation of the human actue phase serum amyloid A genes, SAA1 and SAA, J. IMMUNOL. 169:399-406 (2002). Uhlar, C. M., and Whitehead, A. S., Serum amyloid A, the major vertebrate acute-phase reactant, EUR. J. BIOCHEM. 265:501-523 (1999). Urieli-Shoval, S., Cohen, P., Eisenberg, S., and Matzner, Y., Widespread expression of serum amyloid A in histologically normal human tissue. Predominant localization to the epithelium, J. HISTOCHEM. CYTOCHEM. 46:1377-1384 (1998). Yamazaki et al., BIOCHEMICAL AND BIOPHYSICAL RES. COMM., 290:1114-1122 (2002). Yankner et al., SCIENCE 250: 279-282 (1990) Zhang et al., NEUROSCI. LETT. 312: 125-128 (2001)
131369DNAhomo sapiens 1atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgggtgtcag cagccgaagc 60ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct 120gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg ggggaactat 180gatgctgcca aaaggggacc tgggggtgtc tgggctgcag aagcgatcag cgatgccaga 240gagaatatcc agagattctt tggccatggt gcggaggact cgctggctga tcaggctgcc 300aatgaatggg gcaggagtgg caaagacccc aatcacttcc gacctgctgg cctgcctgag 360aaatactga 3692122PRThomo sapiens 2Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg65 70 75 80Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 1203570DNAhomo sapiens 3agggacccgc agctcagcta cagcacagat cagcaccatg aagcttctca cgggcctggt 60tttctgctcc ttggtcctga gtgtcagcag ccgaagcttc ttttcgttcc ttggcgaggc 120ttttgatggg gctcgggaca tgtggagagc ctactctgac atgagagaag ccaattacat 180cggctcagac aaatacttcc atgctcgggg gaactatgat gctgccaaaa ggggacctgg 240gggtgcctgg gccgcagaag tgatcagcaa tgccagagag aatatccaga gactcacagg 300ccatggtgcg gaggactcgc tggccgatca ggctgccaat aaatggggca ggagtggcag 360agaccccaat cacttccgac ctgctggcct gcctgagaaa tactgagctt cctcttcact 420ctgctctcag gagacctggc tatgaggccc tcggggcagg gatacaaagt tagtgaggtc 480tatgtccaga gaagctgaga tatggcatat aataggcatc taataaatgc ttaagaggtc 540aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5704122PRThomo sapiens 4Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 12054286DNAhomo sapiens 5gatggttgac aactcccctc ctcttccccc tcttctactg tctactcctg ggaccaagtg 60agccacgcca gctcagatac tacactgacc acagggaatc ccaccttttc caaggaatgg 120aagttgtgta gggaatattc aaatgttgct tagcattgcc ttagataaga accaaaggga 180cagggaaatc ctctgacagc tatctgcctt ataactttca ttttactgtg cctaaaatat 240gctcagaacc cagaaagagg cataattcct aattttggca ggctctaatc taaaataatg 300attctcaaac atggtgtgac ttttgtctat ttgctttatc ctgggtcact gctcctcttc 360tgtcagatac tgggattcca atgagacaaa tggaaatgga gacgtagacc ctctgacctt 420ctatctttta tctatacaca tacacctgtg tgtgtgtgtg tgtgtgtgtg tgtgcgtgtg 480taaaaccgag tgggtttttt tcttggaatg aaagaatgga ctaacattac aaaaaataaa 540aacttgaaac agaatgtgta ttatccttgg ttgtgtttcc ttggccctgc agcaggatga 600agctctccac tggcatcatt ttctgctccc tggtcctggg tgtcagcagc caaggatggt 660taacattcct caaggcagct ggccaaggtg aggtccacag gatagggggc aggaggctgc 720ttctggctgc ccccaggatg cagctgagca gaggccacat ccccactggg caaaggtgct 780agtgatgcca cagatggata gagaaggggc atggtttttc ataagcgtgg ttcctcatgc 840ttttctggac agctttgaca ctcttctatg aggatcctcc agccgaggtc gcataaggtg 900tgagctgcct cttttcagca ggaccatgag agagatgtgg agttgagggg tgcatgttcc 960cataataccg gtggggctct actgccccct agtgggaaat ctgggacagt tcatgtctat 1020gtctcctggg aagccaggaa gcaggtggat caaaagtgtg aggcgagtcc atggggaagc 1080tgaacggagc caaccgtccc cataaaaaca accaagctta gctgagattt taatacgtac 1140taggcactgt ttaaatgtac taatgaattg gtttccatca tttagtccta tgatgcaagc 1200agcattatcc cttaacagag aagctaacac acacacacac acacacacac taacacacac 1260acacacacac acacacacac aaaccccaag atacgtaaag aagttccaaa gcagagcagg 1320attaacccag gcagtcttgc tctgcagaac ttgctcttaa tcaaggtact ctgctgcttt 1380caaaacaaga gtttcggatt tgtgaacaca tagctcatcc tttatctaag aaatggcaaa 1440taggatgtgg tgcctttgga aggtaagtct agctccactt atcccagtaa aacctacagt 1500gaattacctt gatggtggtt ctactggggc ttatatatgg ccaggaaact gctagcaaga 1560gaaatatacc ccgagggctg ggcacagtgg ctcacacctg taatcccagc actttgggag 1620gctgaggtgg gcagatcacc tgaggtcaag agttcgagac cagcctggcc aacatggcga 1680aatcctgtct ctactaaaaa tacagaaatt agccgggtgt ggtggcatgc gcctataatc 1740ccagcctctc gggaggctga gggagaagaa ttgcttgaac tcaggaggca gaggttgcag 1800tgagctgtga tcacaccact gcactccagc ctaggagaca gagcaagact ccatctagag 1860agacagagag agagagagag ggagaaatat accccactag ccataataaa gtggcaaaat 1920tttgttttca gaatgcagta ttttaaattt caggtattat tatttttctg agtctctgaa 1980aaatggtttt aaggatttgc ttttaatcct atttacatgt tcacacactc aactacaaat 2040atctttcatt ccttaggtta atatttttca aagggttgtt ctgggaccac ttgcgtgaga 2100atcacctgga ttctgggatg ctttgtgaaa tgaaatgaag attcccgggt ccatacccta 2160ccccctgccc ccaacagcca cagtctcttg ggacagagcc tagaaatctt gcctttgcta 2220agcacctcgg tagattttta tgcacagcaa aggttgagaa ccactacctc ttgttttgct 2280gctgaaagtg ataaaatgtg ccaggaattt tggaagtact tattaagcca atctgaacat 2340caaggagcca tttaagtcag taactcagag gaataagtag agtaaaaatg tcataaactc 2400tcaataaaag caatcaattt aacaccagga gtaataaatg cataaaatga agatgagtta 2460tctaatagag aaattatata aaccatgatt ataactctat atttgagttc ccccttttcc 2520gtaatcagtt aattttctaa aaaatcttcg tcacttaatt ctagcttgat cagatccctt 2580cagtccgtaa ctccctgctc ctcatcttag tttagccctt cttttttctt atgccacctt 2640tcctaaggac cagagaagtg aaatgataat atattggcca cctacaatgt tctagacatc 2700atacatgtat tttctctgct cttctgcata atcactgtga ggcaggcaat actcctccat 2760ttcattgggg aggacattga ggttctgaac tagtgggtca gttgtccttt ttctgaattt 2820gattacccag tagtataaag ctttcttagg taactcacct ttatcacttg ctgactgaat 2880tctgacagat gtcagtttct aattatagcc tggacattca gatgtattca ggaccaagtt 2940gtcctcactc tacctacagg catgaatttc tctcattgac taggttagga gcgccatatg 3000tctgcagcct ccctcagaat cccctgtgtt ctcacaccag ggaactgagg gttccctggg 3060tccttccagg tagaagttca ttgtacaatg aaacatccct taaggaccat ttcatctctt 3120ctttaggtgc atcacacatg gttaaaacaa agtaataaca gaacttagaa tggaatcaaa 3180cagaatgaaa cttacaccaa gtacaattct cattacatta acccagagaa gtgaaaagta 3240gaagaatatt tatttcaagc caatataatt tccaagggct ttgttgaagg ctgaaatctt 3300cgggaggaaa gtagtgagaa gaaaactgtt cattcctcta ttttcccagt atataattgt 3360tttgatcatt ttctttcctt tccagggact aaagacatgt ggaaagccta ctctgacatg 3420aaagaagcca attacaaaaa attcagacaa atacttccat gcttggggga actatgatgc 3480tgtacaaagg gggcttgggg ctgtctgggc tacagaagtg atcaggtaat gcacattcct 3540gatgttgcca ggaatgagtg agcagagctt gactgccttg gacagtcagg agagaggtaa 3600gctccttgca gagaagttag aggctgcagc ccctcctcct cttgccctct ctctgcctgt 3660gtgcttagtg cgagggtctg agtggatggt agaagtgagt gattcctcac cctccctctc 3720tgggtgctgt tcatccagcc taggggtgcc cagcctggct gagtggggca gtgcccaggc 3780agggtcattg ttttcacccc tccttccttg gccttcctgg gcttctccca gagtcctccc 3840ttggaaagca gagaatggga aggtgggctg ttgctcactg gcctggtgat taatctcctt 3900gcttgcctgg actacagcga tgccagagag aacgtccaga gactcacagg agaccatgca 3960gaggattcgc tggctggcca ggctaccaac aaatggggcc agagtggcaa agaccccaat 4020cacttccgac ctgctggcct gccagagaaa tactgagctt ccttttcaat ctgctctcag 4080gagacctggc tgtgagcccc tgagggcagg gacatttgtt gacctacagt tactgaattc 4140tatatcccta gtacttgata tagaacacat aaaaatgctt aataaatgct tgtgaaatcc 4200agtttgttat tggaatctgg aagcagaata tgacagtctt cctgggatca tgggcctgtt 4260tagtaccata gggatgacca ataaac 42866193DNAhomo sapiens 6gttttctgct ccttggtcct gggtgtcagc agccgaagct tcttttcgtt ccttggcgag 60gcttttgatg gggctcggga catgtggaga gcctactctg acatgagaga agccaattac 120atcggctcag acaaatactt ccatgctcgg gggaactatg atgctgccaa aaggggacct 180gggggtctgg gct 193764PRThomo sapiens 7Val Phe Cys Ser Leu Val Leu Gly Val Ser Ser Arg Ser Phe Phe Ser1 5 10 15Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg Asp Met Trp Arg Ala Tyr 20 25 30Ser Asp Met Arg Glu Ala Asn Tyr Ile Gly Ser Asp Lys Tyr Phe His 35 40 45Ala Arg Gly Asn Tyr Asp Ala Ala Lys Arg Gly Pro Gly Gly Leu Gly 50 55 608369DNAhomo sapiens 8atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgagtgtcag cagccgaagc 60ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct 120gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg ggggaactat 180gatgctgcca aaaggggacc tgggggtgcc tgggccgcag aagtgatcag caatgccaga 240gagaatatcc agagactcac aggccatggt gcggaggact cgctggccga tcaggctgcc 300aataaatggg gcaggagtgg cagagacccc aatcacttcc gacctgctgg cctgcctgag 360aaatactga 3699122PRThomo sapiens 9Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 12010369DNAhomo sapiens 10atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgagtgtcag cagccgaagc 60ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct 120gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg ggggaactat 180gatgctgcca aaaggggacc tgggggtgcc tgggccgcag aagtgatcag caatgccaga 240gagaatatcc agagactcac aggccgtggt gcggaggact cgctggccga tcaggctgcc 300aataaatggg gcaggagtgg cagagacccc aatcacttcc gacctgctgg cctgcctgag 360aaatactga 36911122PRThomo sapiens 11Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln Arg Leu Thr Gly Arg Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 1201210001DNAhomo sapiens 12gggtggatca cgaggtcagg agatcgagac catcttggct aacatggtga aaccccgtct 60ctactaaaaa tacaaaaaaa ttagccgggc gtcatggtgg gcgcctgtag tcccagctac 120tcgggaggct gaggcaggag aatggtgtga acccgggagg cagaacttgc agtgagccta 180gatcgcgcca ctgcactcca gcctggggga caaaacgaga ctctgtctca aaaaaaaaaa 240aaaaaattcc cacattagag ttggggaaat gggcagtcct ggtggaagtt agggaacaga 300tctgggacac gttatagcca gctggactac aggaggccat aagctcaatt cttccttgac 360tctgaaacct tccactggtc ctaatgccta gtaattccag gcctttccca gttgtgccag 420gcttggaggt gaacacatct atgtgccaag aaggaaaggt atgccaagca ggggcttaag 480tcatccttat cctcagtctg tctatgagtg gtatgtaccc ctgttcccct tgcaagatct 540gctgggctta ggtctcctgg ctgtgagttc cccatacctg ggcataaatg tagtgagcct 600gagctcccaa ataaggttgg gggctccaga gaggtggaga gccctgtgtc tgggaagtgt 660gcccacccag caggtctgac caggaagata cactgctagg gttatggaaa aagactatgt 720gtcaaggtct cttgattctc catctaggca gagaatcatc tttaattaat gggaaactgg 780aaggcaaatt acttggacct gaaattactt tttgtttatt gaaccactgt gttgtaaatc 840acatctctct gaaggcaaga gaaatcaggg agttacaaaa tgtttaggag aactaaacag 900gactccctgt tttgctaact aatcagattg agacaggctc tctggtaaat ctacaaattt 960gatgttgttc aaccataagc agtaaatttc ctatgctgga ttttcctgac aatgaatgta 1020aaaggaaaag gagtcttttt gacaaaatat tttattgttc atctaaactg aaaaacttct 1080ctatttttca aaattgctat acgtgtttaa agatgtagat atttgaatag cctaactggt 1140acagaaggtt taatgatgat tcctaagaca tacctataaa ttacttgaaa ttgaaacgaa 1200atttaagaag aattattgga attttcccct tctcaaatga gttcttagtt tcataaatac 1260tatacaagtc cataagagat ttggggtttt gagatgtctt tttttttttt tttttttcag 1320acggagtttc actgttgttg cctaggctgg agtgcaatgg cgtgacctca gctcactaca 1380acctccacct cccaggttca agcgattttc ctgcctcagc ctcccaagta gctgggatta 1440cagggacctg ccacaacgcc aagctaatgt tttgtatttt tagtagagat ggggttcacc 1500atgttggcca ggcttgtctg gaactcctga cctcaggtga tccacccgcc tataatttat 1560tactcccttt tgcaaatgtt tgaaaaggaa taaagtgcaa tatttttaaa cagaatgcag 1620agttctgttg tcctttggca ataccagttt cagactctga gagtggctct tgctgttgcc 1680gacagtgggc tgatgaccaa atcccaacat gcccccgctg cgagtccttc ataacctgat 1740tcagtcatca cttagaggcc agcaggcttc agggaggcgt gagcctcagc caacaaccta 1800taggggaaga gacgcagaac tcaatgcaga caggtttgga ttctggtgcc tagagaatgc 1860aacttggaaa ctctgagcca ggagaaaagg gttctctctc catgagagag tgtgggcttt 1920gtgagaagcg acacacagca aacacaatta agagtccacc cctcagcggg gcgcaggggc 1980tcacgcctgt aatcccagca ctttgggagg ccgaggcggg tggatcacga ggtcaggaga 2040tcaagaccat cctggctaac acagtgaaac cctgtctcta ctaaaaatac aaaaaaatta 2100gccgggcgtg gtggcgggcg cctgtggtcc cagctactcg ggaggctgag gcaggagaat 2160ggtgtgaacc cgggaggtgg agcttgcagt gagccgagat cgcgccactg cactccagcc 2220tgggcgacag agcgagactc catctcaaaa aaaaaaagaa aaagaaaaag aaaaagagtc 2280cgcccctgaa ttaaatagtt ggtccttttg tgttcctggt gattcacttg ctaagtggaa 2340gaaacaggag ggaatctttt ctcctgccct cctggtaatc catagcccat ggcctggctt 2400tacttctgta aagtggcagg agaccttttg acagctgagc catttcttat tttatttatt 2460ttaataagag atggtaggaa tgagcaatga tattagtacc tggggactgt tgttcttaag 2520gagaaacaat cttagaatga ttagtgatac cccttgcttt ctcttttctt tcattatact 2580ttttgtacac atatttttcc catttattta ttggaatctt actgatttat tataagtata 2640agctttatgt ctacacatgt ataatcattt ttccccaagt ataagtctct ttttcatgga 2700ggcacagcct agacctggtt agccgccatc tcccctcatt gtatgcccaa tatctattgt 2760agtatctgct gcatagaagg cactcgatgc gtgaatggat aatgactgat gatgaatcaa 2820taaataaatg gacatgtcat tgtaaaaaat tctaaaaatc tagaataaca caagctgttg 2880gcactaccta gaaacacaga tgtaaaactt cctaggttgt gtttcaccat gggaacatgt 2940ctttgaacaa aaatgggatc atattctatt gcactctttc ccttaagaga tacttctcca 3000ggtcattaag tgctcttcca caatatcagt atatggcaga ggcaaggtca taccaggtct 3060gtctgaaacc agggcttggc tcttaacttg cagccatact gcctccaagt ctaggtggct 3120gggttttagg atctgtaatg ggaactcagt gtcacaacct ctactgggaa ggtattctgg 3180tgttgcataa caggactttc tgttagagat aaccatggca aaatggaata gagacaaagt 3240tcaggtttct gctgccagga gctgagattg ctgtgaccaa tggcattctc ccaaaccaaa 3300taatccaacc tggaattacc ataaaccact cctcatcttt tcaaggggtg tccaagttcc 3360cagaaaagaa catttgttaa gggatggagg caaggaggtg gagaagaaag agcactggcc 3420aaggtatcat gagtgtcctg ggttctggtc cttgaataag ccatttatct tctctgcagc 3480ttctccatct gataggagtt tggaggcaga gttttttctt aatgagcaaa agacagtcgt 3540gcctaggaga tgtggtgtac atgttagaaa gaagggactg gctgtgactc tataaaagat 3600gaattcatac aaaaacaaat taccctttcc cagggagaaa gtttggatcc agtaattaga 3660gatctcaaaa agtagaagac ctgccctgtg aggcctgtgg cctccaagtt tgaatgctgt 3720gtgtcagctt taaaaactag tttcttgctg ataaatgttt catattaagc atgtgttgag 3780agtactcctt gcctaccttc actagccact gtttccttcc cctcctccct tgtcccttca 3840ttctctccag aactttctgc taacttccat tctcttcagg acttcagcat ggttgggaga 3900agatcagaaa ggcatcctca ctgtttttat tttagtccac ttgacctttg gggagtagtt 3960ccactggctc ataagtatca gccccccata gcacagcacc ccacactgag cccggaagca 4020ataaagaatc ccaatctgct gtcactaacc agcacgctca actgccatgc cctttactct 4080tctcatctcc ctgctttcac gtcacaccaa ctaatttctc tatgagtcag cctcaactct 4140cccaacactc tgcccaccct tcttctacta ccttccagtg agctcctcga aagaagggtc 4200tgcggtgagg atgccccttt atctctgcct atttccttcc cattacaaaa acttgaaacc 4260tgcctttccc atgttgattt cactttattc tcatctttac ccatggggta tgcctcctgc 4320aattcctcct agacaataga atgagaaaga ggggtcctcg tcctctttgc tttccatgac 4380catttctcca ttcttcacct ctgtgatgtg tcctctttga agtccctgat aaattcatta 4440ccaccttctc tccagtctta ctaatgttat ctgcacaagt gatttccaaa caggaagatt 4500ttcaaacact gattcctgaa gatcaccccc aactcgctga actgagacca agacctccaa 4560gattatggct taggaatctg catttttttt ttttttttga gacaagagtc tcgctctgtt 4620gccaggctag agtgcaatgg tggaatcata gctcattgta acctcaaact cctgggctca 4680agtgatcttc ctgcctcagc ctcccaagta gtgaggacaa caggagtgtg ccaccatgcc 4740cagctaattg ttaatttttt gtagaaatgg agtctcacta tgttgctcgg gctggtctca 4800aactcctgac cttaacccat cctccgcctc cgcccccaaa agtgttggga ttacaggtgt 4860gagccaccgt gcccagccta gaaataccca ctagaagctt ctgtgtagac aatctgctta 4920gtgatgtttg gagacaaagt acctctttat tgtattcatt gacaaaactc tccagtcctc 4980tcccatcttc atggaaaatt ttcacagttc atttacggcc ctctttccaa cacattcact 5040gccaatactc ttattgacaa taactgtatt gttgaacctt
ccagtatcct gcattcccgg 5100atcaaggccc cctcaaagcc ctgatatgca aatatctggg aaaagaatgt tccagaggaa 5160aggaacagct aatccgaggc ccctagggta agatgtgcct gggggtttgg agaccagtgt 5220ggccagagca aaatgagcag gaggagagaa ttggatgatg aggtacgaga ggaaggagtt 5280aggacagttt gagtaaagtt tgaaaaccat tataagggct ttgacttcaa ctatgagtgg 5340aagtggaatc ctccggagag ttttgaatgg agagtgatag aagttgtctt gtgttgtaac 5400agtctggctg ctatactgaa aagagactag ttggcggcaa agggggaaat gtggaagcca 5460gttaagaagc catcataacc cagaaggtga tgcctaataa catctctctg ggagcagcgg 5520agagatgata agggtttgcc ttctgaatat gttttttgac aattaatgta aacatttcaa 5580gtaggctgag attttattgc atattaacaa tgtccatgtt cactcgcggc agccgccccc 5640ttctgcgcgg tcatgccgag ccagcacctg ggcctggaac tgggccgcag cccccagctt 5700cacccaccac ctccctacca tggacccctg caaagtgaac gagcttcggg cctttgtgaa 5760aatgtgtaag caggatccga gcgttctgca caccgaggaa atgcgcttcc tgagagagtg 5820ggtggagagc atgggaggta aagtaccacc tgctactcag aaggctaaat cagaagaaaa 5880taccaaggaa gaaaaacctg atagtaagaa ggtggaggaa gacttaaagg cagacgaacc 5940atcaactgag gaaagtgatc tagaaattga taaagaaggt gtgattgaac cagacactga 6000tgctcctcaa gaaatgggag atgaaaatgt ggagataacg gaggagatga tggatcaggc 6060aaatgataaa aaagtggctg ctattgaagt cctaaatgat ggtgaactcc agaaagccat 6120tgacttattc acagatgcca tcaagctgaa tcctcgcttg gccattttgt atgcaaagag 6180ggccagtgtc ttcgtcaaat tacagaagcc aaatgctgcc atccaagact gtgacagagc 6240cattgaaata aatcctgatt cagctcagcc ttacaagtgg cgggggaaag cacacagact 6300tctaggccac tgggaagaag cagcccatga tcttgccttt gcctgtaaat tggattatga 6360tgaagatgct agtgcaatgc tgaaagaagt tcaacctagg gcacagaaaa ttgcagaaca 6420ttggagaaag tatgagcgaa aacatgaaga gcgagagatc aaagaaagaa tagaacgagt 6480taagaaggct caagaagagc aggagagagc ccagagggag gaagaagcca gacgacagtc 6540aggagctcac tatggccctt ttccaggtgg ctttcctggt ggaatgcctg gtaattttcc 6600cggaggaatg cctggaatgg gaggggacat gcctggaatg gccggaatgc ctggactcaa 6660tgaaattctt agtgatccag aggctcttgc agccatgcag gatccagaag ttatggtggc 6720cttccaggat gtggctcaga acccagcaaa tatgtcaaaa taccagagca acccaaaggt 6780tatgaatctc atcagtaaat tgtcagccaa atttggaggt caagcataat gcccttctga 6840taaataaagc cctgctgaag gaaaagcaac ctagatcacc ttatggatgt cgcaataata 6900caaaccaacg tacctctgac cttctcatca agagagctgg ggtgctttga agataatccc 6960tacccctctc ccccaaatgc agctgaagca ttttacagtg gtttgccatt agggtattca 7020ttcagataat gttttcctac taggaattac aaactttaaa cactttttaa atcttcaaat 7080atttaaaaca aatttaaagg gtctgttaat tcttatattt ttctttacta atcattgtgg 7140atttttcctt aaattattgg gcagggaata tacttattta tggaagatta ctgctctaat 7200ttgagtgaaa taaaagttat tagtgcgagg caaacataaa aaaaaaaagt ccatgttcat 7260ctctaaatga catcattgtt ccaaagcttt tccattcttc ttaaccttcc acctgtcaat 7320ctataggaga tgacttctcc tacttcactc atgcattgac tccttcaatc aataaaagtg 7380actaagaacc tgctacaggt gaggtgctgt gtttggtgtt aaagtgacaa cagttatctg 7440tcaataagcc tgacaaggtt cctatccctg tgttttgtgc actctgggtc aaactcagaa 7500atgcaaacag gtggagagcg atgagttcta tgactggtaa agaaaagggc ctgctggttt 7560ccctcaggat ctctgtcctt catctcaaaa tgcatcttcc ttgttatcgt tcctctcctt 7620cctgtctcag aggaagacct gctcctgcta cactctgggc aaccttgtcc ccgtggccct 7680gtggcccctt ggttgttgaa gtctatgtta tgccctatct tttaccctca gtcactctct 7740ctgttaacat tctccctgtg ccctgtaacc ctccctcatc tttaaataaa tcctcctcct 7800ttgaccttcg catgtattca gtcatgcaac tcaacaagca tttattgcac agtgatattc 7860aatttgccac ttgctaaaag tctgaacctt ggcagctgaa tgtgatcaga aaaaaagcac 7920gactgctatg actagtctca ctttaaattc atggtcgttg accaagagct accatacaat 7980ccactacctt tctcaagttc agtcacattc ttcctttcct agatgtctgc tttctacttc 8040tcttctcttc tgaaacttcc cacaactcct cgttcattct cttctcagtt gacaactttg 8100cttcctattt cactgaaaaa tagaagcaat cagatatgaa cttctggctg ggcatggtag 8160ctcatgccta taatctcagc actttgggag gccaaggcag gaggactgca ggttaggaat 8220ttgagaccag cctgggcaac atggtgaaac tcccactgta ctaaaaattt taaaaattac 8280tcaaacatat tggcaaacaa ctgcagtccc agctacttgg gaggttgaga tgcaaggatc 8340acttaaacct gggaggctga ggctgcagtg agccatgatt gcaccactgc actccagctc 8400aggcaacaga gcaagaccct gtcttgagag gagaggagaa gagaggaggg gaggggaggg 8460caggggaggg gaggggaggg gaagggagag gggaggggag aggggaggag agaggggagg 8520ggaggggagg ggaggggagg ggaggagagg aggatcaggt gaggagtatg ccaaggagtg 8580tttttaagac ttactgtttt ctctttccca acaagattgt catttccttt aaaaagtagt 8640tatcctgagg cctatattca tagcattctg aaagaaagaa aagaaaagag gaaagaaaga 8700gagaggaagg aaggaaggag aaagagagag gaaggaagga gaaagagaga ggaaggaagg 8760gaggaagaga agaagggagg aagaaaagaa ggaaggaagg agggagggag ggaagggagg 8820gagggaaaga ggaagaaagg agggaaagaa ggaaggaaga gagagaggaa ggaaggagga 8880agagagaaga aggaaggagg aagacagaga gggagtaagg aaggaaggaa ggagaaagag 8940agaggaagga agaaatgaag gaaggaagga aagaaagaaa aaataaaaga gtgaaaacgg 9000actggagaag aagaaaccac agttgctgct atatccacca gcctctctgc atgtcctggc 9060ctcagccctg ctgggctctg gtactgacca cttccttcct tcctaatttc ctaattgact 9120aggccagctg agcagggctt ttctgtgctg aggaggtaaa tctctggata tctagactga 9180ggggtggaag gagccttcca gggcacacat gagacatggc aggggtaggc tgctagtttt 9240attttgtttt cttttagaca cagggtcttg ctctgttaac caggctggag tgcagtggcg 9300tgattatagc tcactgcagc cttgacctcc tgggtctccc acaatccttc cgcttcagcc 9360tcttgagtag ctgggactgc aggtgcacac taccacaccc ggtccattta tttttatatt 9420tcgtagagac aagatcttac agttttgcac agagtgatct taaactcttg accccaagtg 9480atcctcctgc cttggcctcc aaaagcattg ggattatagg agtgagccac tgtgctggac 9540ctagtctgtc agctttgaag ctttagatat gaactcagag ggacttcatt tcagaggcat 9600ctgccatgtg gcccagcaga gcccatcctg aggaaatgac tggtagagtc aggagctggc 9660ttcaaagctg ccctcacttc acaccttcca gcagcccagg tgccgccatc acggggctcc 9720cactctcaac tccgcagcct cagccccctc aatgctgagg agcagagctg gtctcctgcc 9780ctgacagctg ccaggcacat cttgttccct caggttgcac aactgggata aatgacccgg 9840gatgaagaaa ccactggcat ccaggaactt gtcttagacc gttttgtagg ggaaatgacc 9900tgcagggact ttccccaggg accacatcca gcttttcttc gctcccaaga aaccagcagg 9960gaaggctcag tataaatagc agccaccgct ccctggcagg c 100011310001DNAhomo sapiens 13gtctgccagg gagaggtggc tgctatttat agtgagcctt gctggtctct tgggagggaa 60gaaaagctgg atgtggtccc tggggaaagt ccctgcaggt catttcccct acaaactggt 120ctaagacaag ttcctggatg ccggtggttt cttcatcccg ggtcatttat cccagttgtg 180taacctatgg gaacaagaga ggtttgctgt gccttggcaa tggacagggt gctagatcag 240ctctgctcct cagcattggg ggaagtgcag ctgcagagat gccagtggga gccccgtgat 300ggcggcacct gggctgctgg aaggtgtgga gtgagggcag ctcttcagcc agctcctgac 360tataccggtc atttcctcag gatgggccct gctgggccac atggcagatg accctgactg 420aaatccctgt gagttcatgt ctaaagcttt aagctttaaa acggacagcc tacccctgcc 480acatctcatg tgtgccctgg aagcctcctt ccacccctct ggatgtcctg atatttctca 540gcacagaaaa tctctgctcc gctggcttag ccaatttgga aatgcttttt ctaagttggc 600tcctgagcca aggacaatgt agagaggggg actttctgct gccccagcct agtcctggag 660ccccaccttg ggagaatgag agtgtggtgc gttaaatagg cagcccagct ggggacgtgc 720ccagcatcca ggcagggaag ggtgggagag ctcttggtct gctgtattat cacggagggg 780tgcagggggc atgcagatca ctctctcatg agaacatcaa cagggtcaga ttagctctgc 840agaggcttat ggaggagcat ggtggccaga gatgggtcag taccagagcc caggggggct 900gaggccagga catgcagaga ggctggtgga catagcagca actctggttt cttcttctcc 960agtccatgtt cataccctga gggctaggca tttgtaataa caaacaaaca agcaatttag 1020aaatgggcca ggcatggtgg catgtgccta tagtcccagc tacttgggag gccaaggcag 1080gaggcctgct tgaacccaga aatttgaggc cagcctgggc aacacagcaa gattatctta 1140aaaaattttt tttaatctct gagaaatggg tagggccagg aagtaaagga tggccaaata 1200ctccataagc agcaaatgcg tggctccaat gtgaacaatg atattataga ctctgttctg 1260agacctatgc attgacacct ccacctcccc cactacatct tgccacctta aaaccactga 1320gagtggtacc tgctggaatg ggtccacaca cacagtcaca catattttag gcagggtagt 1380tgacatcccc agggaaaaag agctcacaga gagaggctga atgtttccaa ctgggtagca 1440gtaatagtac atcatgctgt acatggtaca gcacagatca ggtgaaaata atagcacatc 1500gtgattaacc agggcttatt ccagggagtc aagaagagtt tcatatcaga aaaatctatc 1560tttgtaattc actataccag taatcaaaga aaaggattgt acatttattt tactagatgc 1620agaaaatgaa tttcataatt gtcaacatct actgatgata aggaaaatgt ataacaaaat 1680aaagagacca tttctgactt gagaaaggat aaataccaat atgttatagc aacagttctc 1740aaactgtttt ccagggaacc ctaagaatcc ctccttaggg aggctttgat ctcaaaatta 1800tttttagaat agtgctaaca cactattttc atgtttcagt ctcattttct catgagtaca 1860cacaatatga caagttagtt gatatgagtg tggatttcca catggtaact gacttttcag 1920aagctaccac ttgttgagtt tggtataata tagaatagcc acaattatct aaaaatacca 1980ttaaaataca ctcccccatt tcaactatat atctgtgtga ggctgaattt tcttcatata 2040ctccaaccta aataacatat taaaacaggt tggatgatga atcagatagg aaaatccagc 2100tatgaaaaaa aaatcagaca tgaaaaattt tcaaaagggt aaaaccatag tactcttctt 2160actttttttc ttttggaaga tggttatttt tcataaaaat atattattta tgttaacata 2220tagaagatgg ataatttttt gaagaattga taaatgttta aattttttct ttctattatg 2280gtaaatactg atgaatagag tccccataaa taaaagttct ttggggtatt caataatttt 2340taatagtgta atgggatcct gagaccaaaa ggtttgagaa tcattgctct acagcaaaca 2400ttatgtgtaa ttaagacact tcaggtgcat tctcaagaag accaataaag aggccacaat 2460ggcaggcgtg gtggctcaca cttgtaatcc aagaacttag agaggacgag gcaggtggat 2520cactggaggt caggaattct caaccagcct ggccaacatg gtgaaaccct gtctctacta 2580aaagtacaaa aattagtcgg gtgtagtggc aggtacctgt aatcccaagt acttgggggg 2640ttgaggcagg agaatcactt gaagccggga ggtggaggct gcagtgagcc gagatcgtgc 2700cactgcactc cagcctgggc aacggagtga gacttcatca tggaaaaaaa aacaaagagg 2760ccaggatgtc tggttgttac tgccactgtt tcacatatcc ctgaaggacc tgcccaatgc 2820taaagaaaca caaggaaggt aagaggtgaa agagaagaaa tgaaactatc attgtttgaa 2880gatgacacca tcttttacat agaaaacctg ttagaatcaa atggcaagct attagaacta 2940ctaagagaat tcagtgaggc tgctgtattc atggcaaaat tttaacaatt gatagcattt 3000ctctgcaaca ttccttaata gttataaaat acagcacaaa gtagtaccaa aaatattaac 3060tatctaggaa ataacctctt acagagaaaa tttagtctgt taaaggataa acagtggcaa 3120tgtacgtcat gtccacagag attatatttt agcttagcaa agataccaat tctcccaaat 3180ttatttataa attaaatgca atgtgaatca aaatttccca ctggaatttt tatcaggaag 3240gcaacaaatt ctttctttct ttctttcttt ctttctttat ttatttattt atttatttat 3300ttatttattt ccttccttcc ttccttcctt ccttccttcc tttctttctt tctttctttc 3360tttctttctt tctttctctc tctctttctc tctccccccc tctctctctc tctgtctctc 3420tctctctctc tttctttctt tctttctttc tttcttttta agacaaagtc tggctctgtc 3480acccaggctg cagtgcagtg atacaatctc agctcactga aacctcaacc tctccggcat 3540caggtgaacc tcccacctca gccccccgag tagctgggac tacaggtgca caccactggg 3600cctagataac tttttgtatt tattgtaaat aaacacaaaa aataaatatt ttgctcaggt 3660tggtctggaa ctcctgggct caagcaatcc gcctgccttg gcctcccaaa gtgctagaat 3720tacagttgtg agccaccaca cccagccaat aaattaattc tttatgatga ataagttatc 3780tatgaaaatt aagtcagctg ggtgcggtgg ctcacgcctg taatcccagc actttgccgg 3840gctgaagcag gtggatcacc tgaggttggg agttcaagac cagccggacc aacatagaga 3900aaacccgtct ctactaaaaa tgcaaaatta gctgggtgtg gtggcatatg cctgtaatcc 3960cagatactta ggaggctgag gcaggagaat tgcttgaacc cgggcggtgg aggttgcggt 4020gagccaagat tgcaccattg cactccagcc tgggccacaa gagcgaaact ccatctcaaa 4080aaaaaaaaaa gagaagttaa gtcaatgaaa agttaagtca attaaaaaag taagagctgt 4140agtgtttaga tatatacaca cacacatata tatatattta tctttatata tgtatatata 4200tcttttcctt tttttgagac cgagtctgtt tttgttgccc aggctggaat gcagtggcgc 4260gatctctgct tactgcaacc tctgcctccc aggttcaagc gattctcgtg cctcagcctc 4320ccgagtagct gggattacag gtgcctgccc ccatgcccgg ctaatttttg catttttagt 4380agagacgggg tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caggtgatcc 4440accggcctca gcctcccaaa gtgctgggat tacaggtgtg agccaccgcg cccagccata 4500tattttgctt ttcatctgca gctcctggat cctaactcct tgttatattg ttgggcactt 4560taggcctcag taaacagaat ctctgtctat gaccttctcc tgtccttctt ccacctgccc 4620aaagcaggac tctaatttga ttgtgggtca aaagactctc attccagaaa gggccttgcc 4680tcatacccta gaggaaggaa tgctgcacag aaacgccaag tctgaacaga caagccttgc 4740tgggtttata ccatatgctt tttgtccaat cacatttctt catggttgcc aatcatgcct 4800atgtaatgaa gcctccataa gaacccagaa ggacagggtt cagagagttt ccacatagct 4860gaacactatc tggagagtga acacttccta gagagtggca cacccagaga gatcatgaaa 4920gctccacgcc cctttcccct tacctcgccc tccacatctc ttcatctgta tctttcataa 4980tatcctttat aaataaacca gcaaatgtgt ttccctgagt tatgtgagtc actctagcaa 5040attaatcgaa cccaaagagg gggtcatggg aaccccaact tgaagccagt cagtcagaag 5100ttccagaggc ccagacttgc aactggggag aaagaggggg aggtcttggg gactgagccc 5160ccaacctgtg ggatctgaca ctgtctccag gtaggtagtg ttggaactgc attggaggac 5220actcctggtg tctgctgctt ggtgtgtggg gggaaaaacc cacacctttg gttacggagg 5280tcttctgtgt tgacgatcat tgctgtttga gggcagaggg aatacacggt ttgagagagt 5340ttttccctga catgagcgaa caggggacat gtactggtct ctgagatggg ggatcatggg 5400atctgccaca agtggggaga ccactgtgac ccctgccaca gtctttgggg cagagggtgt 5460ctcgggggca gaagaagcga gagttgtttg cagtagcagt tatgtccaaa gtgggcgcca 5520ggaaagtagg gctgcccagc tttgaagagc ctccttactc ccagcctgaa tgaaaccatt 5580tcctgtaaag cgctaagcat aaagtttgcc aatggtgatc cacggagaag tgagtgtacc 5640ccaccccgcc atcccacagg gaatgtcgga gtgatgttga tctgcaccta gggaaggaat 5700ggttcatgag atgtggtgga gatgctgagg gcccgtggac atcagatcct accctacctg 5760tgccaggaca agccatgcgc atgtgcttca gaccaccagg caacaggagt gttgcatgag 5820gtgtgaagca ggcacctggg aaagaggagt gtgaacagca gatgggacac actgggggca 5880gtcataggaa tgaaatgtcc caggatggat gcaggcaggt tatggaggac ttagtgagga 5940ctgctctcct ggtgggaatt gtggagtggg agactggatg gagactggag gtgttttaag 6000tagggaagcc aacttgcaag ggtgaccagg gaaactatgt cggccaaggg tgagacatgc 6060actggcaaga ctctcagaca gcctggctta tctaagcaga atgcttgagc catgccaacg 6120gtgcctcgca agttgtatta atcatgtcct ttcattttgt gtttttggtg cttggcatct 6180gggcccttgc tgaccctaag ggaccatttc tctcagagct agtcaagtcc tagacacagt 6240aaatgactct cctgggagca tgccttccat gtgcagacca accaatcaag agtccacact 6300cccacccacc tcctttatcg agctctcaca tcctggggca ccatccacct gccctaatca 6360ctcaaggacc acgtcccaaa caactaggga cagcctccat gcccctgcac ccattgaaat 6420tattcatgct agccaatcct aaacctgtgt atgctgccac accattcctt cctgcagaaa 6480cacagtaagg actcttccta cacctcccct acttcctctg ctccctgact tacccactta 6540cttcctggtg cagtcccctg tggcatagtt cactctcttc ttttgggaac tgtgaggcta 6600tcttctcaat ggcagtcatc tcctgagctg ttggccttgc catacctaac taataataaa 6660atctatattc taaggtaaaa acaaaacaga tagggtctca ctctgttgcc caggctggag 6720tacagtggtg tgatcatgac tcactgcagc ctcaaactcc tgggctcaag cagttctctc 6780atctcaacct cccgagtagc tgggactaca ggcacacacc accatgcctg gctagttttc 6840ttattttttt tgtagataca gggtcttgtt atgttgccaa ggctggtctt gaactcctgg 6900gctcaagtga tcctcctgcc ttggcctccc aaactgctgc aattacaggc atgagccacc 6960atgcccagat cagaaatctt actaaaaata tttcaaggag aagagaaagc caaagatgtt 7020gaatatatat atatgtgtgt gtgtgtgtgt gtatatatat gtatatatgt gtatatatgt 7080gtgtatatat atatgtatat atgtatatat atatgtatat atgtatatat atatgtatat 7140tggggcaggc gtggtggctc atgcctgtgg tcctaactac ttgagagtct gaggtgggag 7200gattgcttga gcctgggaga tcgaggctgc tgtgagctga gactacacca ctgcactcca 7260gcttgggtga cagagtgaga ccctgtctcc aaaaaaacaa aaagaaaaag aaaaaaagat 7320ggaaaaagac atgaaaaaac aacaacagaa atacccacac atcatcaatg ggagggaagc 7380atcttgaggc agcaaagcgg gagtgctagt agagaggcag atagggcgtt ggacctgagg 7440cattaaggaa agtcaggatt tggagcttac aagtctctca ttggagatgg gatggggttg 7500gaatgaatgt ctgagcaaac acaaagcatt tccttcccta atgactcccc accagtctaa 7560agaatcccac attaggtcga acacggtggc tcacgcctgt aatcccagca ctttgggagg 7620ccaaggcggg tggatcacga ggtcaggaga tcgagaccat cttggctaac atggtgaaac 7680cccgtctcta ctaaaaatac aaaaaaatta gccgggcgtc atggtgggcg cctgtagtcc 7740cagctactcg ggaggctgag gcaggagaat ggtgtgaacc cgggaggcag aacttgcagt 7800gagcctagat cgcgccactg cactccagcc tgggggacaa aacgagactc tgtctcaaaa 7860aaaaaaaaaa aaattcccac attagagttg gggaaatggg cagtcctggt ggaagttagg 7920gaacagatct gggacacgtt atagccagct ggactacagg aggccataag ctcaattctt 7980ccttgactct gaaaccttcc actggtccta atgcctagta attccaggcc tttcccagtt 8040gtgccaggct tggaggtgaa cacatctatg tgccaagaag gaaaggtatg ccaagcaggg 8100gcttaagtca tccttatcct cagtctgtct atgagtggta tgtacccctg ttccccttgc 8160aagatctgct gggcttaggt ctcctggctg tgagttcccc atacctgggc ataaatgtag 8220tgagcctgag ctcccaaata aggttggggg ctccagagag gtggagagcc ctgtgtctgg 8280gaagtgtgcc cacccagcag gtctgaccag gaagatacac tgctagggtt atggaaaaag 8340actatgtgtc aaggtctctt gattctccat ctaggcagag aatcatcttt aattaatggg 8400aaactggaag gcaaattact tggacctgaa attacttttt gtttattgaa ccactgtgtt 8460gtaaatcaca tctctctgaa ggcaagagaa atcagggagt tacaaaatgt ttaggagaac 8520taaacaggac tccctgtttt gctaactaat cagattgaga caggctctct ggtaaatcta 8580caaatttgat gttgttcaac cataagcagt aaatttccta tgctggattt tcctgacaat 8640gaatgtaaaa ggaaaaggag tctttttgac aaaatatttt attgttcatc taaactgaaa 8700aacttctcta tttttcaaaa ttgctatacg tgtttaaaga tgtagatatt tgaatagcct 8760aactggtaca gaaggtttaa tgatgattcc taagacatac ctataaatta cttgaaattg 8820aaacgaaatt taagaagaat tattggaatt ttccccttct caaatgagtt cttagtttca 8880taaatactat acaagtccat aagagatttg gggttttgag atgtcttttt tttttttttt 8940ttttcagacg gagtttcact gttgttgcct aggctggagt gcaatggcgt gacctcagct 9000cactacaacc tccacctccc aggttcaagc gattttcctg cctcagcctc ccaagtagct 9060gggattacag ggacctgcca caacgccaag ctaatgtttt gtatttttag tagagatggg 9120gttcaccatg ttggccaggc ttgtctggaa ctcctgacct caggtgatcc acccgcctat 9180aatttattac tcccttttgc aaatgtttga aaaggaataa agtgcaatat ttttaaacag 9240aatgcagagt tctgttgtcc tttggcaata ccagtttcag actctgagag tggctcttgc 9300tgttgccgac agtgggctga tgaccaaatc ccaacatgcc cccgctgcga gtccttcata 9360acctgattca gtcatcactt agaggccagc aggcttcagg gaggcgtgag cctcagccaa 9420caacctatag gggaagagac gcagaactca atgcagacag gtttggattc tggtgcctag 9480agaatgcaac ttggaaactc tgagccagga gaaaagggtt ctctctccat gagagagtgt 9540gggctttgtg agaagcgaca cacagcaaac acaattaaga gtccacccct cagcggggcg 9600caggggctca cgcctgtaat cccagcactt tgggaggccg aggcgggtgg atcacgaggt 9660caggagatca agaccatcct ggctaacaca gtgaaaccct gtctctacta aaaatacaaa 9720aaaattagcc gggcgtggtg gcgggcgcct gtggtcccag ctactcggga ggctgaggca 9780ggagaatggt gtgaacccgg gaggtggagc ttgcagtgag ccgagatcgc gccactgcac 9840tccagcctgg gcgacagagc gagactccat ctcaaaaaaa aaaagaaaaa gaaaaagaaa 9900aagagtccgc ccctgaatta aatagttggt ccttttgtgt tcctggtgat tcacttgcta 9960agtggaagaa acaggaggga atcttttctc ctgccctcct g 10001
Patent applications by Abbot F. Clark, Arlington, TX US
Patent applications by Loretta Graves Mcnatt, Hurst, TX US
Patent applications by Wan-Heng Wang, Fort Worth, TX US
Patent applications by ALCON, INC.
Patent applications in class The ring nitrogens are bonded directly to each other (e.g., pyridazine, etc.)
Patent applications in all subclasses The ring nitrogens are bonded directly to each other (e.g., pyridazine, etc.)