Use of serum amyloid a gene in diagnosis

Tag : lead tetraoxide


Abstract
The present invention provides compositions and methods fortreating glaucoma, methods for diagnosing glaucoma, and methods foridentifying agents which may be useful in the treatment ofglaucoma. More specifically, the present invention describes theuse of agents that modulate the expression of serum amyloid A.
Description of the Invention
SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of theprior art by providing methods to diagnose and compositions totreat glaucoma. In one aspect, the present invention provides amethod for treating glaucoma by administering to a patient in needthereof a therapeutically effective amount of a compositioncomprising an agent that interacts with a gene encoding serumamyloid A protein (SAA), or with the gene's promoter sequence. Theinteraction between the agent and the gene encoding SAA, or withits promoter sequence, modulates the expression of SAA, such thatthe patient's glaucomatous condition is treated. In preferredembodiments, the agent will be a protein, peptide, peptidomimetic,small molecule or nucleic acid.

In another aspect, the present invention provides a method fortreating glaucoma by administering to a patient in need thereof atherapeutically effective amount of a composition comprising anagent that inhibits interaction of the serum amyloid A protein(SAA) with its receptor. Preferably, the agent will be a peroxisomeproliferator-activated receptor .alpha. (PPAR.alpha.) agonists,tachykinin peptides and their non-peptide analogs or .alpha.-lipoicacid. Most preferably, the agent will be fenofibrate, Wy-14643,(4-chloro-6-(2,3-xylidino)-2-pryrimidinylthiol)-acetic acid),ciprofibrate, 2-bromohexadecanoic acid, bezafibrate andciglitizone, bafilomycin, concanamycin or pseudolaric acid B.

The present invention further provides a pharmaceutical compositionfor treating glaucoma comprising a therapeutically effective amountof a serum amyloid A protein (SAA) antagonist and a pharmaceuticalcarrier. The antagonist contained in the composition may be any ofthe compounds identified above.

In yet another embodiment, the present invention provides a methodfor diagnosing glaucoma, by the following steps: a) obtaining abiological sample from a patient; and b) analyzing said sample foran aberrant level, aberrant bioactivity or mutations of the geneencoding serum amyloid A protein (SAA) or its promoter region orits gene products, wherein said gene encoding SAA comprises thesequence set forth in SEQ ID NO:1 or SEQ ID NO:3, wherein itspromoter region comprises the sequence set forth in SEQ ID NO:12 orSEQ ID NO:13, and wherein SAA comprises the sequence set forth inSEQ ID NO:2 or SEQ ID NO:4; wherein the aberrantly high level,aberrantly high bioactivity or mutations of the SAA genes or thegene products indicates a diagnosis of glaucoma.

In preferred aspects, the biological sample is ocular tissue,tears, aqueous humor, cerebrospinal fluid, nasal or cheek swab orserum. Most preferably, the biological sample comprises trabecularmeshwork cells.

Alternatively, the present invention provides a method fordiagnosing glaucoma in is a patient, by the steps: a) collectingcells from a patient; b) isolating nucleic acid from the cells; c)contacting the sample with one or more primers which specificallyhybridize 5' and 3' to at least one allele of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:12, or SEQ ID NO:13 under conditions such thathybridization and amplification of the allele occurs; and d)detecting the amplification product; wherein aberrant level ormutations of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:12, or SEQ IDNO:13, in the sample indicates a diagnosis of glaucoma.

The present invention also provides a method for identifying agentspotentially useful for treating glaucoma, by the steps: a)obtaining cells expressing SAA (SEQ ID NO:1 or SEQ ID NO:2) orcells containing SAA promoter/reporter gene such that the reportergene is expressed; b) admixing a candidate substance with thecells; and c) determining the level of SAA protein (SEQ ID NO:2 orSEQ ID NO:4) or the level of gene expression in the cells;

wherein an increase or decrease of the production of SAA protein orgene expression in the presence of said candidate substanceindicates an agent potentially useful for the treatment ofglaucoma.

In another aspect, the present invention provides a method foridentifying an agent potentially useful for treating glaucoma, bythe steps: a) forming a reaction mixture comprising: (i) an SAAprotein or a cell expressing SAA or a reporter gene driven by anSAA promoter; (ii) an SAA protein binding partner; and (iii) a testcompound; and b) detecting interaction of the SAA protein andbinding partner or level of reporter gene products in the presenceof the test compound and in the absence of the test compound;

wherein a decrease or increase in the interaction of the SAAprotein with its binding partner in the presence of the testcompound relative to the interaction in the absence of the testcompound indicates a potentially useful agent for treatingglaucoma.

In another aspect, the present invention provides a method foridentifying an agent potentially useful for treating glaucoma, bythe steps: a) forming a reaction mixture comprising: (i) cellscomprising SAA recombinant protein (SEQ ID NO:2 or SEQ ID NO:4) orcells comprising expression vectors comprising SEQ ID NO:1 or SEQID NO:3; and (ii) a test compound; and b) detecting the effect ondownstream signalling (IL-8) in the presence of the test compoundand in the absence of the test compound; wherein a decrease orincrease in the downstream signalling in the presence of the testcompound relative to the interaction in the absense of the testcompound indicates a potentially useful agent for treatingglaucoma.

In preferred aspects, the cells containing the SAA protein orexpression vectors will be HL-60 cells.

DETAILED DESCRIPTION PREFERRED EMBODIMENTS

Glaucoma is a heterogeneous group of optic neuropathies that sharecertain clinical features. The loss of vision in glaucoma is due tothe selective death of retinal ganglion cells in the neural retinathat is clinically diagnosed by characteristic changes in thevisual field, nerve fiber layer defects, and a progressive cuppingof the ONH. One of the main risk factors for the development ofglaucoma is the presence of ocular hypertension (elevatedintraocular pressure, IOP). IOP also appears to be involved in thepathogenesis of normal tension glaucoma where patients have what isoften considered to be normal IOP. The elevated IOP associated withglaucoma is due to elevated aqueous humor outflow resistance in thetrabecular meshwork (TM), a small specialized tissue located in theiris-corneal angle of the ocular anterior chamber. Glaucomatouschanges to the TM include a loss in TM cells and the deposition andaccumulation of extracellular debris including proteinaceousplaque-like material. In addition, there are also changes thatoccur in the glaucomatous optic nerve head (ONH). In glaucomatouseyes, there are morphological and mobility changes in ONH glialcells. In response to elevated IOP and/or transient ischemicinsults, there is a change in the composition of the ONHextracellular matrix and alterations in the glial cell and retinalganglion cell axon morphologies.

The present inventors have discovered that the expression of SerumAmyloid A (SAA) mRNA and protein are significantly upregulated inglaucomatous TM tissues and cells. The inventors have verified thedifferential mRNA expression seen using Affymetrix gene chips byreal time quantitative polymerase chain reaction (QPCR) andincreased SAA protein levels by SAA ELISA. This is the first timeSAA has been shown to be expressed in the TM.

Human SAA comprises a number of small, differentially expressedapolipoproteins encoded by genes localized on the short arm ofchromosome 11. There are four isoforms of SAAs. SAA1 (SEQ ID NO:2),encoded by SEQ ID NO:1, and SAA2 (SEQ ID NO:4), encoded by SEQ IDNO:3, are known as acute phase reactants, like C-reactive protein,that is, they are dramatically upregulated by proinflammatorycytokines. The 5'UTR promoter regions of SAA1 and SAA2 genes arealso 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 levelconstitutively expressed gene encoding constitutive SAA4 (SEQ IDNO:7). SAA2 has two isoforms, SAA2.alpha. (SEQ ID NO:9), encoded bySEQ ID NO:8, and SAA2.beta. (SEQ ID NO:11), encoded by SEQ IDNO:10, which differ by only one amino acid. SAA1 and SAA2 proteinsare 93.5% identical at the amino acid level (SEQ ID NO:2 and SEQ IDNO:4, respectively) and these genes are 96.7% identical at thenucleotide level (SEQ ID NO:1 and SEQ ID NO:3, respectively).

SAA is an acute-phase reactant whose level in the blood is elevatedapproximately is 1000-fold as part of the body's responses tovarious injuries, including trauma, infection, inflammation, andneoplasia. As an acute-phase reactant, the liver has beenconsidered to be the primary site of expression. However,extrahepatic SAA expression was described initially in mousetissues, and later in cells of human atherosclerotic lesions(O'Hara et al. 2000). Subsequently, SAA mRNA was found widelyexpressed in many histologically normal human tissues. Localizedexpression was noted in a variety of tissues, including breast,stomach, small and large intestine, prostate, lung, pancreas,kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, andbrain neurons. Expression was also observed in lymphocytes, plasmacells, and endothelial cells. SAA protein expression co-localizedwith SAA mRNA expression has also been reported in histologicallynormal human extrahepatic tissues. (Liang et al. 1997;Urieli-Shoval et al. 1998).

SAA isoforms are apolipoproteins that become a major component ofhigh-density lipoprotein (HDL) in the blood plasma of mammals anddisplaces A-I (ApoA-I) and phospholipid from the HDL particles(Miida et al. 1999). SAA binds cholesterol and may serve as atransient cholesterol-binding protein. In addition, over-expressionof SAA1 or SAA2 leads to the formation of linear fibrils in amyloiddeposits, 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. SAAconcentration 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 withwhich to monitor inflammatory disease activity. Hepaticbiosynthesis of SAA is up-regulated by pro-inflammatory cytokines,leading to an acute phase response. Chronically elevated SAAconcentrations are a prerequisite for the pathogenesis of secondaryamyloidosis, a progressive and sometimes fatal diseasecharacterized by the deposition in major organs of insolubleplaques composed principally of proteolytically cleaved SAA. Thissame process also may lead to atherosclerosis. There is arequirement for both positive and negative SAA control mechanismsto maintain homeostasis. These mechanisms permit the rapidinduction of SAA expression to fulfill host-protective functions,but they also must ensure that SAA expression is rapidly returnedto baseline levels to prevent amyloidosis. These mechanisms includemodulation of promoter activity involving, for example, the inducernuclear factor kB (NF-kB) and its inhibitor IkB, up-regulation oftranscription factors of the nuclear factor for interleukin-6(NF-IL6) family, and transcriptional repressors such as yin andyang 1 (YY1). Post-transcriptional modulation involving changes inmRNA stability and translation efficiency permit further up- anddown-regulatory control of SAA protein synthesis to be achieved. Inthe later stages of the AP response, SAA expression is effectivelydown-regulated via the increased production of cytokine antagonistssuch as the interleukin-1 receptor antagonist (IL-1Ra) and ofsoluble cytokine receptors, resulting in less signal transductiondriven by pro-inflammatory cytokines (Jensen and Whitehead 1998).

There are several reports suggesting that primary amyloidosis maybe associated with glaucoma. For example, it was found that amyloidwas deposited in various ocular tissues including the vitreous,retina, choroid, iris, lens, and TM in primary systemic amyloidosispatients (Schwartz et al. 1982). Ermilov et al. (1993) reportedthat in 478 eyes of 313 patients, aged 25 years to 90 years, withcataracts, glaucoma, and/or diabetes mellitus, 66 (14%) of the eyescontained amyloid-pseudoexfoliative amyloid (PEA). Krasnov et al.(1996) reported that 44.4% of 115 patients with open-angle glaucomarevealed extracellular depositions of amyloid. Amyloidosis wasrevealed in the sclera in 82% of the cases and in the iris in 70%of the cases. A number of clinical conditions, includingAlzheimer's disease, exhibit aberrant amyloid tissue depositsassociated with disease. However, amyloids are molecularlyheterogeneous and encoded by different amyloid genes. The previousreports are unclear regarding which amyloid(s) might be associatedwith glaucoma. The present inventors have shown, for the firsttime, that SAA gene expression is elevated significantly inglaucomatous TM tissues. Increased SAA may be involved in thegeneration of elevated IOP and damage to the optic nerve leading tovision loss in glaucoma patients. The present invention providesmethods of using a finding of increased SAA expression to diagnoseglaucoma. The present invention further provides methods forscreening for agents that alter SAA expression or function in orderto identify potentially anti-glaucomatous agents. In anotheraspect, the present invention provides methods and compositions ofusing agents that antagonize SAA actions and/or interactions withother proteins for the treatment of glaucoma.

Diagnosing Glaucoma

Based on the inventors' finding that certain subjects with glaucomahave increased levels of SAA expression, the present inventionprovides a variety of methods for diagnosing glaucoma. Certainmethods of the invention can detect mutations in nucleic acidsequences that result in inappropriately high levels of SAAprotein. These diagnostics can be developed based on the knownnucleic acid sequence of human SAA, or the encoded amino acidsequence (see Miller 2001). Other methods can be developed based onthe genomic sequence of human SAA or of the sequence of genes thatregulate expression of SAA. Still other methods can be developedbased upon a change in the level of SAA gene expression at the mRNAlevel.

In alternative embodiments, the methods of the invention can detectthe activity or level of SAA signaling proteins or genes encodingSAA signaling proteins. For example, methods can be developed thatdetect inappropriately low SAA signaling activity, including forexample, mutations that result in inappropriate functioning of SAAsignaling components, including SAA induction of IL-8. In addition,non-nucleic acid based techniques may be used to detect alterationin the amount or specific activity of any of these SAA signalingproteins.

A variety of means are currently available to the skilled artisanfor detecting aberrant levels or activities of genes and geneproducts. These methods are well known by and have become routinefor the skilled artisan. For example, many methods are availablefor detecting specific alleles at human polymorphic loci. Thepreferred method for detecting a specific polymorphic allele willdepend, in part, upon the molecular nature of the polymorphism. Thevarious allelic forms of the polymorphic locus may differ by asingle base-pair of the DNA. Such single nucleotide polymorphisms(or SNPs) are major contributors to genetic variation, comprisingsome 80% of all known polymorphisms, and their density in the humangenome is estimated to be on average 1 per 1,000 base pairs. Avariety of methods are available for detecting the presence of aparticular single nucleotide polymorphic allele in an individual.Advancements in the field have provided accurate, easy, andinexpensive 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; Syvanenet al. 1990; Kuppuswamy et al. 1991; Prezant et al. 1992; Ugozzoliet al. 1992; Nyren et al. 1993; Roest et al. 1993; and van derLuijt et al. 1994).

Any cell type or tissue may be utilized to obtain nucleic acidsamples for use in the diagnostics described herein. In a preferredembodiment, the DNA sample is obtained from a bodily fluid, e.g.,blood, obtained by known techniques (e.g. venipuncture), or buccalcells. Most preferably, the samples for use in the methods of thepresent 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 upontissue sections (fixed and/or frozen) of patient tissue obtainedfrom biopsies or resections, such that no nucleic acid purificationis necessary. Nucleic acid reagents may be used as probes and/orprimers for such in situ procedures (see, for example, Nuovo 1992).

In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, forexample, by utilizing a differential display procedure, Northernanalysis and/or RT-PCR.

A preferred detection method is allele specific hybridization usingprobes overlapping a region of at least one allele of an SAAsignaling component that is indicative of glaucoma and having about5, 10, 20, 25 or 30 contiguous nucleotides around the mutation orpolymorphic region. In a preferred embodiment of the invention,several probes capable of hybridizing specifically to other allelicvariants involved in glaucoma are attached to a solid phasesupport, e.g., a "chip" (which can hold up to about250,000 oligonucleotides). Oligonucleotides can be bound to a solidsupport by a variety of processes, including lithography. Mutationdetection analysis using these chips comprising oligonucleotides,also termed "DNA probe arrays" is described e.g., inCronin et al. (1996). In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. Thesolid phase support is then contacted with a test nucleic acid andhybridication to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

These techniques may further include the step of amplifying thenucleic acid before analysis. Amplification techniques are known tothose of skill in the art and include, but are not limited to,cloning, polymerase chain reaction (PCR), polymerase chain reactionof specific alleles (ASA), ligase chain reaction (LCR), nestedpolymerase chain reaction, self sustained sequence replication(Guatelli et al. 1990), transcriptional amplification system (Kwohet 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 sizeanalysis, detecting specific tagged oligonucleotide primers in thereaction 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 aplurality of markers simultaneously. For example, it is well knownin the art to select PCR primers to generate PCR products that donot overlap in size and can be analyzed simultaneously.Alternatively, it is possible to amplify different markers withprimers that are differentially labeled and thus can each bedifferentially detected. Of course, hybridization based detectionmeans allow the differential detection of multiple PCR products ina sample. Other techniques are known in the art to allow multiplexanalyses of a plurality of markers.

In a merely illustrative embodiment, the method includes the stepsof (i) collecting a sample of cells from a patient, (ii) isolatingnucleic acid (e.g., genomic, mRNA or both) from the cells of thesample, (iii) contacting the nucleic acid sample with one or moreprimers which specifically hybridize 5' and 3' to at least oneallele of SAA that is indicative of glaucoma under conditions suchthat hybridization and amplification of the allele occurs, and (iv)detecting the amplification product. These detection schemes areespecially useful for the detection of nucleic acid molecules ifsuch molecules are present in very low numbers.

In a preferred embodiment of the subject assay, aberrant levels oractivities of SAA that are indicative of glaucoma are identified byalterations in restriction enzyme cleavage patterns. For example,sample and control DNA is isolated, amplified (optionally),digested with one or more restriction endonucleases, and fragmentlength sizes are determined by gel electrophoresis.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the allele.Exemplary sequencing reactions include those based on techniquesdeveloped my Maxim and Gilbert (1977) or Sanger (1977). It is alsocontemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays,including sequencing by mass spectrometry (see, for exampleWO94/16101; Cohen et al. 1996; Griffin et al. 1993). It will beevident to one of skill in the art that, for certain embodiments,the occurrence of only one, two or three of the nucleic acid basesneed 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 anuclease, hydroxylamin or osmium tetraoxide and with piperidine)can be used to detect mismatched bases in RNA/RNA or RNA/DNA orDNA/DNA heteroduplexes (Myers et al. 1985b; Cotton et al. 1988;Saleeba et al. 1992). In a preferred embodiment, the control DNA orRNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employsone or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called "DNA mismatch repair"enzymes). For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cellscleaves T and G/T mismatches (Hsu et al. 1994; U.S. Pat. No.5,459,039).

In other embodiments, alterations in electrophoretic mobility willbe used to identify aberrant levels or activities of SAA that areindicative of glaucoma. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences inelectrophoretic 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 inpolyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.1985a). In a further embodiment, a temperature gradient is used inplace of a denaturing agent gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner 1987).

Examples of other techniques for detecting alleles include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutationor nucleotide difference (e.g., in allelic variants) is placedcentrally and then hybridized to target DNA under conditions whichpermit hybridization only if a perfect match is found (Saiki et al.1986; Saiki et al. 1989). Such allele specific oligonucleotidehybridization techniques may be used to test one mutation orpolymorphic region per reaction when oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations or polymorphic regions when the oligonucleotides areattached to the hybridizing membrane and hybridized with labeledtarget DNA.

Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunctionwith the instant invention. Oligonucleotides used as primers forspecific amplification may carry the mutation or polymorphic regionof interest in the center of the molecule (so that amplificationdepends on differential hybridization) (Gibbs et al. 1989) or atthe extreme 3' end of one primer where, under appropriateconditions, mismatch can prevent, or reduce polymerase extension(Prossner 1993). In addition it may be desirable to introduce anovel restriction site in the region of the mutation to createcleavage-based detection (Gasparini et al. 1992). It is anticipatedthat in certain embodiments amplification may also be performedusing Taq ligase for amplification (Barany 1991). In such cases,ligation will occur only if there is a perfect match at the 3' endof the 5' sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence orabsence of amplification.

In another embodiment, identification of an allelic variant iscarried out using an oligonucleotide ligation assay (OLA), asdescribed, E.g., in U.S. Pat. No. 4,998,617 and in Landegren et al.1988). Nickerson et al. have described a nucleic acid detectionassay that combines attributes of PCR and OLA (Nickerson et al.1990). In this method, PCR is used to achieve the exponentialamplification of target DNA, which is then detected using OLA.

Several techniques based on this OLA method have been developed andcan be used to detect aberrant levels or activities of SAA that areindicative of glaucoma. For example, U.S. Pat. No. 5,593,826 andTobe et al. (1996), describe such techniques that are frequentlyused.

In one embodiment, fenofibrate, a peroxisome proliferator-activatedreceptor .alpha. (PPAR.alpha.) agonist, may be formulated in apharmaceutically acceptable composition and used to treat glaucomaby modulating SAA expression. Studies have shown that fenofibrateand WY 14643 treatment reduces plasma SAA concentration (Yamazakiet al. 2002). It is believed that other PPAR.alpha. agonists, suchas ciprofibrate, 2-bromohexadecanoic acid, bezafibrate,ciprofibrate and ciglitizone may also be useful for the treatmentof glaucoma.

The present inventors further postulate that agents that preventamyloid-induced cell death may be useful for protecting TM andother 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 varioustypes of ophthalmic formulations for delivery to the eye (e.g.,topically, intracamerally, or via an implant). The Compounds arepreferably incorporated into topical ophthalmic formulations fordelivery to the eye. The Compounds may be combined withophthalmologically acceptable preservatives, surfactants, viscosityenhancers, penetration enhancers, buffers, sodium chloride, andwater to form an aqueous, sterile ophthalmic suspension orsolution. Ophthalmic solution formulations may be prepared bydissolving a Compound in a physiologically acceptable isotonicaqueous buffer. Further, the ophthalmic solution may include anophthalmologically acceptable surfactant to assist in dissolvingthe Compound. Furthermore, the ophthalmic solution may contain anagent to increase viscosity, such as, hydroxymethylcellulose,hydroxyethylcellulose, hydroxypropylmethylcellulose,methylcellulose, polyvinylpyrrolidone, or the like, to improve theretention of the formulation in the conjunctival sac. Gellingagents can also be used, including, but not limited to, gellan andxanthan gum. In order to prepare sterile ophthalmic ointmentformulations, the active ingredient is combined with a preservativein an appropriate vehicle, such as, mineral oil, liquid lanolin, orwhite petrolatum. Sterile ophthalmic gel formulations may beprepared by suspending the Compound in a hydrophilic base preparedfrom the combination of, for example, carbopol-974, or the like,according to the published formulations for analogous ophthalmicpreparations; preservatives and tonicity agents can beincorporated.

The Compounds are preferably formulated as topical ophthalmicsuspensions or solutions, with a pH of about 4 to 8. Theestablishment of a specific dosage regimen for each individual isleft to the discretion of the clinicians. The Compounds willnormally be contained in these formulations in an amount 0.01% to5% by weight, but preferably in an amount of 0.05% to 2% and mostpreferably in an amount 0.1 to 1.0% by weight. The dosage form maybe a solution, suspension microemulsion. Thus, for topicalpresentation 1 to 2 drops of these formulations would be deliveredto the surface of the eye 1 to 4 times per day according to thediscretion of a skilled clinician.

The Compounds can also be used in combination with other agents fortreating glaucoma, such as, but not limited to, .beta.-blockers,prostaglandins, carbonic anhydrase inhibitors, .alpha..sub.2agonists, miotics, and neuroprotectants.

Claim 1 of 1 Claim
1. A method for treating glaucoma, said method comprisingadministering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising a small molecule agentthat interacts with a gene encoding serum amyloid A protein (SAA),wherein said small molecule agent is a peroxisomeproliferator-activated receptor .alpha.(PPARa) agonist selectedfrom the group consisting of fenofibrate, WY-14643, ciprofibrate,2-bromohexadecanoic acid, bezafibrate, and ciglitizone, whereinsaid interaction modulates the expression of SAA, and wherein adecrease in expression of SAA treats glaucoma.
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