Link:  Pharm/Biotech Resources

Title:  Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions

United States Patent:  6,864,239

Issued:  March 8, 2005

Inventors:  Peri; Krishna G. (Montreal, CA); Moffett; Serge (St-Laurent, CA); Abran; Daniel (Vaudreuil-Dorion, CA)

Assignee:  Theratechnologies Inc. (St. Laurent, CA)

Appl. No.:  401397

Filed:  March 27, 2003

Abstract

A GPCR-like protein is described, as well as inhibitory/antagonistic compounds and compositions comprising such inhibitors/antagonists of the protein. Such compounds may be used for treating elevated intraocular pressure and conditions associated with elevated intraocular pressure, such as glaucoma.

SUMMARY OF THE INVENTION

The invention relates to R-14 nucleic acids and polypeptides and compounds capable of lowering intraocular pressure and uses thereof.

Accordingly, in an aspect, the invention provides a substantially pure peptide compound of Formula I:

Z₁ -X₁ -X₂ -X₃ -X₄ -X₅ -X₆ -X₇ -X₈ -Z₂ I

wherein:

X₁ is selected from the group consisting of Phe, His, Ile and related amino acids;

X₂ is selected from the group consisting of Ser, Ile, Phe and related amino acids

X₃ is selected from the group consisting of Leu, Ile, Asp and related amino acids

X₄ is selected from the group consisting of Thr, Cys, Ser and related amino acids possessing side chains containing sulfhydryl, hydroxyl or H-bond forming groups;

X₅ is selected from the group consisting of Gln, Ser, Thr and related amino acids;

X₆ is selected from the group consisting of Lys, Pro, Glu and related amino acids;

X₇ is selected from the group consisting of Tyr, Leu, Cys and related amino acids;

X₈ is selected from the group consisting of Cys, Arg, Trp and related amino acids;

Z₁ is an N-terminal group of the formula H₂ N--, RHN-- or, RRN--;

Z₂ is a C-terminal group of the formula --C(O)OH, --C(O)R, --C(O)OR, --C(O)NHR, --C(O)NRR;

R at each occurrence is independently selected from (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, substituted (C₁ -C₆) alkyl, substituted (C₁ -C₆) alkenyl, or substituted (C₁ -C₆) alkynyl; and

"--" is a covalent linkage.

The invention further provides a substantially pure synthetic peptide compound or recombinant peptide compound, said compound having a domain of Formula II:

-X₁ -X₂ -X₃ -X₄ -X₅ -X₆ -X₇ -X₈ II

wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and "--" are as defined above.

In embodiments, the compound is selected from the group consisting of: FSLTQKYC; HIICSPLR; and IFDSTECW. In embodiments, the domain is selected from the group consisting of: FSLTQKYC; HIICSPLR; and IFDSTECW.

The invention further provides a method of lowering intraocular pressure in a subject, said method comprising administering to said subject an effective amount of the above-mentioned compound.

The invention further provides a method of treating in a subject a condition associated with elevated intraocular pressure, said method comprising administering to said subject an effective amount of the above-mentioned compound.

In embodiments, the methods comprise administering said compound to an eye of said subject.

The invention further provides a pharmaceutical composition comprising the above-mentioned compound in admixture with a pharmaceutically acceptable carrier. In an embodiment, the composition is formulated for administration to an eye of a subject.

The invention further provides a commercial package comprising the above-mentioned compound together with instructions for its use. In embodiments, the use is selected from the group consisting of: (a) lowering intraocular pressure in a subject; and (b) treating a condition associated with elevated intraocular pressure.

The invention further provides an isolated nucleic acid comprising a sequence that encodes the above-mentioned domain. The invention further provides a vector comprising the nucleic acid operably-linked to a transcriptional regulatory element. The invention further provides a host cell comprising the vector.

The invention further provides a method of producing the above-mentioned peptide compound, comprising culturing the above-mentioned host cell under conditions permitting expression of the peptide compound.

The invention further provides an isolated nucleic acid comprising a sequence that encodes a polypeptide comprising at least 278 amino acids of SEQ ID NO:2. In an embodiment, the polypeptide comprises SEQ ID NO:2. In an embodiment, the nucleic acid comprises SEQ ID NO:1 or a sequence substantially identical thereto.

The invention further provides a substantially pure polypeptide comprising at least 278, consecutive amino acids of SEQ ID NO:2. In a further embodiment, the polypeptide comprises SEQ ID NO:2.

The invention further provides a vector comprising the above-mentioned nucleic acid operably linked to a transcriptional regulatory element. The invention further provides a host cell comprising the above-mentioned vector.

The invention further provides a method of producing the above-mentioned polypeptide, comprising culturing the above-mentioned host cell under conditions permitting the expression of the polypeptide.

The invention further provides a method for identifying a compound for: (i) lowering intraocular pressure in a subject; (ii) treating a condition associated with elevated intraocular pressure; or (iii) both (i) and (ii); said method comprising: (a) providing a cell expressing an R-14; (b) contacting the cell with a test compound; and determining whether R-14 activity is decreased in the presence of said test compound, said decrease in activity being an indication that said compound may be useful for: (i) lowering intraocular pressure in a subject; (ii) treating a condition associated with elevated intraocular pressure; or (iii) both (i) and (ii). In an embodiment, said decrease in activity is an indication that said compound is an R-14 antagonist. In an embodiment, R-14 comprises a polypeptide comprising SEQ ID NO:2, an active fragment thereof, or a sequence substantially identical thereto.

The invention further provides a use of the above-mentioned compound for: (a) lowering intraocular pressure in a subject; (b) treating a condition associated with elevated intraocular pressure; or (c) both (a) and (b).

The invention further provides a use of the above-mentioned compound or the above-mentioned composition for the preparation of a medicament. In an embodiment, the medicament is for: (a) lowering intraocular pressure in a subject; (b) treating a condition associated with elevated intraocular pressure; or (c) both (a) and (b).

In an embodiment, the above-mentioned condition is glaucoma.

In an embodiment, the above-mentioned subject is a mammal, in a further embodiment, a human.

In further embodiments, Z₁ is selected from the group consisting of a proton, a sequence of 1-3 amino acids, or a blocking group such as a carbamate group, an acyl group composed of a hydrophobic moiety such as cyclohexyl, phenyl, benzyl, short chain linear and branched alkyl groups of 1-8 carbons. In further embodiments Z₂ is selected from the group consisting of proton, NH₂, 1-3 amino acids as well as arylalkyl amines such as benzylamine, phenylethylamine, phenylpropylamine, and aliphatic amines possessing short chain linear and branched alkyl groups of 1 to 8 carbons.

In another aspect, the invention provides a method for formulating a medicament, the method comprising admixing a compound of the invention with a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of lowering intraocular pressure in a subject, the method comprising inhibiting expression and/or activity of an R-14 protein in the subject. In an embodiment, the R-14 protein comprises a polypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID. NO. 2; and

(b) a polypeptide encoded by a first nucleic acid that is substantially identical to a second nucleic acid capable of encoding the polypeptide of SEQ ID NO. 2. In an embodiment, the second nucleic acid is as set forth in SEQ ID NO. 1.

In an embodiment, the above-noted method comprises administering to the subject an effective amount of an agent capable of modulating R-14 activity. In an embodiment, the agent is a compound of the invention. In a further embodiment, the method comprises administering to the subject a therapeutically-effective dose of the above-mentioned composition. In an embodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of lowering intraocular pressure in a subject, the method comprising administering to the subject an effective amount of a compound of the invention. In an embodiment, the method comprises administering to the subject a therapeutically-effective dose of the above-mentioned composition. In an embodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of preventing or treating in a subject a condition associated with elevated intraocular pressure, the method comprising inhibiting expression and/or activity of an R-14 protein in the subject. In an embodiment, the R-14 protein comprises a polypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID NO. 2; and

(b) a polypeptide encoded by a first nucleic acid that is substantially identical to a second nucleic acid capable of encoding the polypeptide of SEQ ID NO. 2. In an embodiment, the second nucleic acid is as set forth in SEQ ID NO. 1.

In an embodiment, the method comprises administering to the subject an effective amount of an agent capable of modulating R-14 activity. In an embodiment, the agent is a compound of the invention. In a further embodiment, the method comprises administering to the subject a therapeutically-effective dose of the above-mentioned composition. In an embodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of preventing or treating in a subject a condition associated with elevated intraocular pressure, the method comprising administering to the subject an effective amount of a compound of the invention. In a further embodiment, the method comprises administering to the subject a therapeutically-effective dose of the above-mentioned composition. In an embodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a commercial package comprising an agent capable of modulating R-14 activity together with instructions for:

(a) lowering intraocular pressure in a subject;

(b) preventing or treating a condition associated with elevated intraocular pressure; or

(c) both (a) and (b).

In an embodiment, the R-14 protein comprises a polypeptide selected from the group consisting of:

(i) the polypeptide of SEQ ID NO. 2; and

(ii) a polypeptide encoded by a first nucleic acid that is substantially identical to a second nucleic acid capable of encoding the polypeptide of SEQ ID NO. 2.

The invention further provides a method for identifying and/or characterizing a compound for lowering intraocular pressure, the method comprising assaying the activity of an R-14 in the presence of a test compound, to identify a compound that acts as an R-14 antagonist, wherein antagonist activity is indicative that the test compound may be useful for lowering intraocular pressure.

The invention further provides a method for identifying and/or characterizing a compound for lowering intraocular pressure, the method comprising:

(a) contacting a test compound with a host cell expressing a polypeptide selected from the group consisting of:

(i) the polypeptide of SEQ ID NO. 2; and

(ii) a polypeptide encoded by a first nucleic acid that is substantially identical to a second nucleic acid capable of encoding the polypeptide of SEQ ID NO. 2; and

(b) assaying activity of an R-14 in the presence of the test compound, to identify a compound that acts as an R-14 antagonist, wherein antagonist activity is indicative that the test compound may be useful for lowering intraocular pressure. In an embodiment, the compound may be used for the prevention and/or treatment of a condition associated with intraocular pressure.

In an embodiment, the above-mentioned subject is a mammal, in a further embodiment, a human.

In an embodiment, the above-mentioned condition is glaucoma.

DETAILED DESCRIPTION OF THE INVENTION

Identification of Novel GPCRs

Use of low stringency hybridization, differential display, microarrays, subtractive hybridization and other techniques followed by cloning of differentially expressed genes in tissues have disclosed several new cDNAs which could potentially code for new G protein-coupled receptors. Following the elucidation of the draft sequence of the human genome (Venter, J C. et al., Science. 291: 1304-1351 (2001); International Human Genome Sequencing Consortium. Nature. 409: 860-921 (2001)), many GPCR-like sequences have been identified based on their canonical seven transmembrane topology as well as conserved protein motifs (Howard, D A et al., 2001. Trends Pharmacol. Sci. 22(3): 132-140). These unique GPCR-like sequences number .about.1000 of which nonolfactory GPCR sequences are estimated to be .about.616.

Gonzalez et al. (Gonzalez, P. et al., 2000. Invest. Ophthalmol. Vis. Sci. 41: 3678-3673), carried out single pass sequencing of 1060 cDNA clones isolated from human trabecular meshwork, including a sequence having homology to MAS-related G-protein coupled receptor (denoted as HTMI-0025, and corresponding to sequences Hs11_-- 9464 and Hs11_-- 24438 found in the human chromosome 11 working draft sequence).

Dong et al. (Dong, X. et al. 2001. Cell 106:619-632) cloned several mas-related cDNAs from mouse embryos lacking neurogenin gene, with subsequent searching among human genome sequences revealing four related genes (called Hs_mrgX1-4).

When the physiological ligand(s) of a GPCR is not known, then it is termed an orphan GPCR. In order to find ligands of these orphan GCPRs, random libraries containing natural peptides isolated from the tissues or those containing small molecules produced by combinatorial chemistry or from natural sources are screened in cell-based and in vitro assays. Selective modulators of the orphan GPCR can be used to assess its function in physiology and pathophysiology using appropriate animal models. Finding a function of the orphan GPCR is a first step towards analyzing its potential as a new drug target.

The studies described herein relate to a novel GPCR protein expressed in trabecular meshwork, nucleic acids capable of encoding it, and peptide compounds capable of modulating phenomena in ocular tissue, notably intraocular pressure.

In one aspect, the invention provides an isolated a GPCR-like reading frame, which is capable of expressing mRNA and protein, and is associated with a role in ocular outflow in animals. The invention further provides three antagonists of the GPCR which could be used for lowering intraocular pressure, and thus for prevention/treatment of disorders associated with elevated intraocular pressure, such as glaucoma and related conditions.

As described herein, applicants have cloned a genomic DNA fragment of 0.97 Kb in length from a BAC clone and have shown that it contained a single exon encoding a 322 amino acid long protein. This protein is termed as R-14. From in silico analysis, R-14 appears to be an integral membrane protein containing seven prominent transmembrane domains and many protein motifs of G protein-coupled receptors. By employing RT-PCR, R-14 mRNA is shown to be expressed in human trabecular meshwork, but not in another ocular tissue, iris. R-14 reading frame was cloned into mammalian expression vectors and transiently as well as stably expressed in HEK293 cells. Using these cells, a protein of 40 kDa was identified to be encoded by the recombinant R-14 gene. Furthermore, using selective peptidic ligands, applicants have shown that inhibition of R-14 receptor resulted in reduction of basal cyclic AMP synthesis, but not basal phosphoinositide levels, in porcine trabecular meshwork and most importantly, reduction of basal intraocular pressure in rabbits, adult pig eyes and anesthetized piglets. Based on this information, R-14 receptor is identified as a useful target for screening for ocular hypotensive drugs, which may be useful for treatment of conditions associated with elevated intraocular pressure such as glaucoma and related conditions.

Furthermore, compositions containing selective inhibitors of R-14 receptor are described which could be potentially used as ocular hypotensive drugs and glaucoma therapeutics or as lead compounds towards development of such drugs. Bioassays in which host cells (e.g. mammalian cells) containing recombinantly expressed R-14 are described which can be used to screen chemical compound libraries to identify lead compounds for providing R-14 ligands which in turn can be optimized into ocular hypotensive drugs and glaucoma therapeutics.

R14 Nucleic Acids

As noted above, the present invention is based, in part, on the discovery of a human gene, which encodes a human nucleic acid and encoding a protein referred to as "R-14". In silico analysis showed that R-14 could be an integral membrane protein containing seven prominent hydrophobic domains and many signature motifs of a G protein-coupled receptor. The protein and nucleic acid of R-14 show strong homology to human, mouse and rat MAS-related G protein-coupled receptors by BLASTN and BLASTP analyses. A10.96 kb subclone of BAC RP11-206c1 (obtained from Sanger Center, UK) was completely sequenced and determined to contain the entire coding region of R-14 (SEQ ID NO:1). The human R-14 coding sequence is 0.966 kb in size and contains no introns. The 966 bp open reading frame (SEQ ID NO:1) encodes a 322 amino acid polypeptide (SEQ ID NO:2). BLASTN analysis of dBEST data base revealed near identity to the EST termed as HTMI-0025F1 (GenBank Acc.: BE439409) and the nucleic acid sequence is identical to HTMI-0025 (Gonzalez, P. et al., supra) with the exception of two nucleotide changes, A to C leading to Gln to Pro at 661158 (NT_-- 009307.3 (Hs11_-- 9464).

The invention provides an isolated R-14 nucleic acid, homologs thereof, and portions thereof. Preferred nucleic acids have a sequence, which is at least about 60%, 65%, 70%, 75%, 80%, 85% and preferably 90% and 95% homologous with a nucleotide sequence of an R-14 gene, e.g., such as a sequence shown in SEQ ID NO. 1. Preferred nucleic acids are vertebrate R-14 nucleic acids. Particularly preferred vertebrate R-14 nucleic acids are mammalian, in an embodiment, human. In one embodiment, the preferred nucleic acid is a cDNA encoding a polypeptide having at least one bioactivity of the subject R-14 polypeptide.

R14 Polypeptides

The present invention features R-14 polypeptides which can be produced in and isolated from, cells or tissues in which the the polypeptide is naturally expressed, or cells in which R-14 polypeptide is expressed using gene transfer of recombinant R1-4 nucleic acids, either as cell-free extracts or purified membrane fractions. Functional forms of the subject polypeptides can be prepared as purified preparations by using a cloned gene as described herein. Preferred R-14 proteins of the invention have an amino acid sequence which is at least about 60%, 65%, 70%, 75, 80%, 85%, 90%, or 95% identical or homologous to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, an R-14 protein of the present invention is a mammalian R-14 protein. In a particularly preferred embodiment an R-14 protein is set forth as SEQ ID NO:2. It will be understood that certain post-translational modifications, e.g., phosphorylation and the like, can increase the apparent molecular weight of the R-14 protein relative to the unmodified polypeptide chain. Protein isoforms encoded by splice variants of R-14 listed in SEQ ID NO:2 are also within the scope of the present invention. Such isoforms may have additional biological activities from those possessed by the R-14 proteins specified by SEQ ID NO:2.

R14 Polypeptide Fusions, Mutant-Proteins and Homologs Thereof

In one aspect, the invention relates to R-14 polypeptides, either as enriched fractions of cells and tissues, or substantially pure preparations of naturally-occurring or recombinantly-produced or chemically-synthesized polypeptides. An R-14 polypeptide of the invention can comprise a full length protein as set forth in SEQ ID NO:2 or can comprises fusion proteins containing smaller fragments corresponding to one or more particular motifs/domains, or fragments comprising at least about 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids in length. The subject R-14 protein also includes within its scope modified proteins, e.g. proteins in which specific mutations prevent post-translational modification, such as glycosylation, myristylation, palmitylation and phosphorylation of the protein, or which mutations prevent or enhance interaction of the R-14 mutant protein with agonists, antagonists or intracellular proteins involved in signal transduction.

In further embodiments, R-14 polypeptides of the invention may comprise a fragment of the polypeptide of SEQ ID NO:2, as defined by a minimum number of consecutive amino acids thereof. Accordingly, in embodiments the invention relates to an R-14 polypeptide comprising at least 100, 125, 150, 175, 200, 225, 250, 275, 278, 280, 290, 300, 305, 310, 315, 320 consecutive amino acids of SEQ ID NO:2.

In another aspect, the, invention relates to a recombinant expression system for producing an R-14 protein. For expression in cells, tissues and animals, the nucleic acid as set in SEQ. ID NO. 1 was cloned into a mammalian expression vector, in which R-14 nucleic acid was operably linked to a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter (for constitutive expression), sequences required for splicing and transcription termination. Such regulatory sequences in conjunction with a R-14 nucleic acid molecule provided a useful vector for gene expression. People skilled in the art could use similar strategies to express R-14 protein in prokaryotic and eukaryotic host cells transfected with appropriate expression vectors in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing R-14 proteins.

The present invention further pertains to methods of producing the subject R-14 polypeptides. For example, a "host cell" transfected with a nucleic acid "vector" directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur. Suitable media for cell culture are well known in the art. The recombinant R-14 polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptides. In a preferred embodiment, the recombinant R-14 polypeptide is a fusion protein containing a domain which facilitates its purification, such as polyhistidine fusion of R-14 protein.

The invention also provides fusion proteins, e.g., R-14 conjugated to green fluorescent protein or beta arrestin. Such fusion proteins can provide detection of R-14 polypeptides in cells, tissues and organisms. Fusions of green fluorescent protein (GFP) to R-14 protein can be used to locate and follow the dynamics of R-14, such as aggregation, association with other cellular proteins, internalization, trafficking, degradation in endocytotic vesicles, in living or fixed cells. R-14 fusions of GFP and luciferase can be used to study and monitor dimer and oligomer formation, association with other signalling molecules. R-14-G_.alpha. protein fusions can be used to measure GTP binding and hydrolysis by the G protein in response to agonists or antagonists and these methods, known to people skilled in the art, are used to screen and/or test small molecule compound libraries for agonist or antagonist activity. These examples are presented to illustrate, but not to limit the potential fusion partners and their uses in basic and applied scientific studies.

Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of one of the subject R-14 polypeptides, which function in a limited capacity as one of either an R-14 agonist (mimetic) or an R-14 antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein. Homologs of each of the subject R-14 proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which display elevated ligand-independent activity or substantially the same, or merely a subset of the biological activity of the R-14 polypeptide from which it was derived. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to an R-14 receptor.

R14 Activity and Assaying Said Activity

In one aspect, the invention provides methods for identifying a compound that can modulate R-14 "activity". Such a method may entail determining the activity of an R-14 protein in the presence of a test or candidate compound. Such a method may for, example be used to identify an R-14 antagonist, which may be useful for lowering intraocular pressure, and further for treating a condition associated with elevated intraocular pressure, such as glaucoma and related conditions. Various aspects of R-14 activity may be assayed in this regard, as noted herein and in the Examples.

In an embodiment, determining R-14 "activity" entails assaying an interaction between an R-14 polypeptide and an R-14 binding partner, to identify compounds that are capable of interfering with the interaction of R-14 and its binding partner, and thus the test compound may be capable of binding to an R-14 polypeptide. In an embodiment, such a method includes the steps of (a) forming a mixture, which includes: (i) an R-14 polypeptide, (ii) an R-14 binding partner and (iii) a test compound; and (b) detecting interaction of the R-14 polypeptide and the R-14 binding partner or alteration in at least one aspect of R-14 polypeptide "activity". A statistically significant change (potentiation or inhibition) in R-14 activity in the presence of the test compound, relative to that in the absence of the test compound, indicates a potential agonist or antagonist (inhibitor) respectively of R-14 bioactivity for the test compound. The reaction mixture can be a cell-free protein preparation, e.g., a reconstituted protein mixture or a cell lysate or purified cell constituents, or a cultured cell recombinantly expressing the R-14 polypeptide or fragments thereof. People skilled in the art can use such a competitive binding assay to detect the interaction between an R-14 polypeptide and a, R-14 binding partner. In an embodiment, at least one of the R-14 polypeptide and the R-14 binding partner comprises a detectable label, and interaction of the R-14 and R-14 binding partner is quantified by detecting the label in the complex. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

Cell-Free Assays

Cell-free assays can be used to identify compounds which are capable of interacting with an R-14 protein, thereby modify the activity of the R-14 protein. Such a compound can, e.g., modify the structure of, an R-14 protein and thereby affect its activity. Cell-free assays can also be used to identify compounds which modulate the interaction between an R-14 protein and a R-14 binding partner. In a preferred embodiment, cell-free assays for identifying such compounds consist essentially in a reaction mixture containing an R-14 protein, R-14 binding partner and a test compound. A test compound can be, e.g., a derivative of a R-14 polypeptide or R-14 binding partner.

Accordingly, one exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) an R-14 polypeptide, (ii) an R-14 binding partner, and (iii) a test compound; and (b) detecting interaction of the R-14 and the R-14 binding protein. For detection purposes, the binding partner can be labelled with a specific marker such as a radionuclide, or a fluorescent compound or an enzyme. Interaction of a test compound with an R-14 protein or fragment thereof can then be detected by determining the level of the marker label after an incubation step and a washing step. The R-14 polypeptide and R-14 binding partner can be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein. A statistically significant change (potentiation or inhibition) in the interaction of the R-14 and R-14 binding protein in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of R-14 bioactivity for the test compound. Radiolabelled samples are counted and quantified by scintillation spectrophotometry. Binding ligands can be conjugated to enzymes such as acetylcholine esterase and bound R-14-binding partner can be quantified by enzyme assay.

Cell-free assays can also be used to identify compounds which interact with an R-14 protein and modulate an activity of an R-14 protein. Accordingly, in one embodiment, an R-14 protein is contacted with a test compound and the bioactivity of R-14 is monitored. The bioactivity of R-14 protein in cell-free assays include, GTP binding, GTP hydrolysis, Dissociation of G_.quadrature. proteins, adenylate cyclase activation, phospholipase (A2, beta, gamma and D isoforms) activation, phospholipid hydrolysis, cAMP synthesis etc. and the methods of measuring these changes in the bioactivity of R-14 protein are well known to those skilled in the art.

Cell Based Assays

In addition to cell-free assays, such as described above, R-14 proteins as provided by the present invention, facilitate the generation of cell-based assays, e.g., for identifying an agent capable of modulating R-14 activity, such as small molecule agonists or antagonists. Cell based assays can be used, for example, to identify compounds which modulate the bioactivity of R-14 protein, expression of an R-14 gene or those genes that are induced or suppressed in response to increased or decreased bioactivity of R-14 protein. Accordingly, in one embodiment, a cell which is capable of producing R-14 is incubated with a test compound in the presence or absence of a natural or synthetic agonist/antagonist of R-14 and the bioactivity of R-14 is measured. The resultant alterations in the bioactivity of R-14 are compared to control R-14 producing cells which have not been contacted with the test compound. These measurements are used to assess the potency, affinity, action of the test compound towards modulating R-14 bioactivity.

A particular embodiment of the present invention is that cell-free and cell-based assays involving the use of R-14 protein as set forth in SEQ ID NO. 2, could become an integral part of a screening system to evaluate and select small molecules which can be optimized to be used as therapeutics for lowering intraocular pressure and in the treatment of glaucoma.

Kits

The invention further provides kits for use in diagnostics or screening methods for providing R-14 binding partners or glaucoma therapeutics. For example, the kit can comprise (1) a labeled R-14 binding partner and/or (2) cell-free lysates or cellular fractions including membranes isolated from R-14 expressing host cells or R-14-expressing tissues or whole cells expressing R-14 protein naturally or by recombinant DNA methods and/or (3) an agent capable of detecting R-14 protein or mRNA and/or (4) means for determining the amount of R-14 protein, mRNA or bioactivity and the means for comparing the amount of R-14 protein, mRNA or bioactivity in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect R-14 mRNA or protein or its bioactivity. Such a kit can comprise, e.g., one or more nucleic acid probes capable of hybridizing specifically to at least a portion of an R-14 gene or allelic variant thereof, or mutated form thereof.

R14 as a Drug Target in Intraocular Hypertension

Applicants' results described herein indicate that R-14 protein represents a drug target, i.e. with a view to provide R-14 modulators which may be useful for lowering intraocular pressure and thus may be useful for the treatment of conditions associated with elevated intraocular pressure, such as glaucoma and related conditions. In a more preferred embodiment, the invention provides a method by which R-14 gene and protein can be expressed in host cells such as mammalian cells, and the cells be used for small molecule or peptide lead compound discovery in order to provide pharmaceutical compositions useful in treating patients diagnosed with elevated intraocular pressure and related conditions such as glaucoma.

R14 Antagonists, Pharmaceutical Compositions, Uses Thereof

Applicants have further identified and characterized agents (e.g. peptide compounds) capable of modulating R-14 activity, e.g. can act as peptide antagonists of R-14 activity. Accordingly, the invention provides peptide compounds for use in lowering intraocular pressure. In a further embodiment, the invention provides peptide compounds for use in the treatment of a condition associated with elevated intraocular pressure such as glaucoma and related conditions. The invention further provides a method of lowering intraocular pressure in a subject and a method for the treatment of a condition associated with elevated intraocular pressure such as glaucoma and related conditions, the methods comprising administering an effective amount of a peptide/peptide compound of the invention, or a composition comprising a peptide of the invention, to the subject, e.g. a subject in need thereof. In an embodiment, the subject is a mammal, in a further embodiment, a human.

Therefore, in an aspect, the invention provides a substantially pure peptide compound of Formula I:

Z₁ -X₁ -X₂ -X₃ -X₄ -X₅ -X₆ -X₇ -X₈ -Z₂ I

wherein:

X₁ is selected from the group consisting of Phe, His, Ile and related amino acids;

X₂ is selected from the group consisting of Ser, Ile, Phe and related amino acids

X₃ is selected from the group consisting of Leu, Ile, Asp and related amino acids

X₄ is selected from the group consisting of Thr, Cys, Ser and related amino acids possessing side chains containing sulfhydryl, hydroxyl or H-bond forming groups;

X₅ is selected from the group consisting of Gln, Ser, Thr and related amino acids;

X₆ is selected from the group consisting of Lys, Pro, Glu and related amino acids;

X₇ is selected from the group consisting of Tyr, Leu, Cys and related amino acids;

X₈ is selected from the group consisting of Cys, Arg, Trp and related amino acids;

Z₁ is an N-terminal group of the formula H₂ N--, RHN-- or, RRN--;

Z₂ is a C-terminal group of the formula --C(O)OH, --C(O)R, --C(O)OR, --C(O)NHR, --C(O)NRR;

R at each occurrence is independently selected from (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, substituted (C₁ -C₆) alkyl, substituted (C₁ -C₆) alkenyl, or substituted (C₁ -C₆) alkynyl; and

"--" is a covalent linkage.

In a further embodiment, Z₁ is an N-terminal group selected from the group consisting of a proton, a sequence of 1-3 amino acids, or a blocking group such as a carbamate group, an acyl group composed of a hydrophobic moiety such as cyclohexyl, phenyl, benzyl, short chain linear and branched alkyl groups of 1-8 carbons. In a further embodiment, Z₂ is a carboxy-terminal group selected from the group consisting of a proton, NH₂, 1-3 amino acids as well as arylalkyl amines such as benzylamine, phenylethylamine, phenylpropylamine, and aliphatic amines possessing short chain linear and branched alkyl groups of 1 to 8 carbons.

The invention further provides a substantially pure synthetic peptide compound or recombinant peptide compound, said compound having a domain of Formula II:

-X₁ -X₂ -X₃ -X₄ -X₅ -X₆ -X₇ -X₈ - II

wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and "--" are defined as above.

In embodiments, the peptide compound comprises a sequence selected from the group consisting of:

NH₂ -Phe Ser Leu Thr Gln Lys Tyr Cys-OH (FSLTQKYC; SEQ ID NO:3);

NH₂ -His Ile Ile Cys Ser Pro Leu Arg-OH (HIICSPLR; SEQ ID NO:4); and

NH₂ -Ile Phe Asp Ser Thr Gln-Cys Trp-OH (IFDSTECW; SEQ ID NO:5).

The invention further provides an active fragment of the above noted peptides. "Active fragment" refers to a fragment of a peptide of the invention which is capable of modulating the activity of an R-14 protein, wherein said R-14 protein comprises a polypeptide selected from the group consisting of:

(a) the polypeptide of SEQ ID NO. 2; and

(b) a polypeptide encoded by a first nucleic acid that is substantially identical to a second nucleic acid capable of encoding the polypeptide of SEQ ID NO. 2.

The invention further provides derivatives of the above (SEQ ID NOs:3, 4 and 5) which may be synthetic polypeptides containing conservative substitutions of individual amino acids, and peptidomimetics thereof.

The invention further provides a recombinant expression system, vectors and host cells, such as those described above, for the expression/production of a peptide comprising a peptide of the invention (such as those set forth in SEQ ID NOs. 3, 4 and 5), using for example culture media, production, isolation and purification methods such as those described above. Such vectors comprise a nucleic acid sequence capable of encoding such a peptide operably linked to a transcriptional regulatory sequence. In an embodiment, the peptide is a fusion peptide containing a domain which facilitates its purification, such as a polyhistidine domain.

The invention also aims to provide a pharmaceutical composition comprising a peptidic or peptidomimetic compound with a pharmaceutically acceptable carrier, wherein said compound is capable of modulating, in an embodiment inhibiting at least one aspect of R-14 polypeptide bioactivity.

The invention also aims to provide a pharmaceutical composition containing a peptidic or peptidomimetic compound with a pharmaceutically acceptable carrier, wherein said compound is capable of decreasing intraocular pressure.

Also within the scope of the invention are pharmaceutical compositions for treating patients diagnosed with increased intraocular pressure and/or glaucoma, comprising administering (e.g., either locally or systemically) to a subject, a pharmaceutically effective amount of a composition comprising a compound capable of modulating at least one aspect of R-14 bioactivity.

R14 Antagonists, Active Fragments, Peptidomimetics Thereof

The invention also provides for reduction of the fragments of R-14 antagonists to generate mimetics, e.g., peptide or non-peptide agents, such as small molecules, which are agonistic or antagonistic of R-14 protein activity.

In order to improve the R-14 antagonists described in this invention for therapeutic use, several modifications of the peptide can be made by substituting a first amino acid with a "related amino acid" which is a second amino acid related to the first amino acid by either structure or function of the side chain: aromatic, aliphatic, positively- or negatively-charged. Examples of related amino acids are provided in Tables 2 and 3 below.

Alternatively, another group of substitutions of the R14 antagonists of the present invention are those in which at least one amino acid residue has been removed and a different residue inserted in its place according to the following Table 2. Another group of substitutions are defined herein as exchanges within one of the following five groups:

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than alpha-helical. Pro, because of its unusual geometry, tightly constrains the chain. It generally tends to promote beta turn-like structures. Cys is capable of participating in disulfide bond formation. Tyr, because of its hydrogen bonding potential, has significant kinship with Ser, and Thr, etc.

In addition, any amino acid representing a component of the said peptides can be replaced by the same amino acid but of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D-amino acid but which can additionally be referred to as the R- or the S-, depending upon its composition and chemical configuration. Additional variations include b- and g-amino acids that provide different spatial arrangement of chemical groups.

In addition to the substitutions outlined above, synthetic amino acids that provide similar side chain functionality can be introduced in to the peptide. For example, aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or L-Phenylglycine, D- or L-2-thienylalanine, D- or L-1-, 2-, 3- or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylalanine D- or L-p-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylalanines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, iso-pentyl groups. Non-carboxylate amino acids can be made to possess negative charge, such as the non-limiting examples of phosphono- or sulfated (e.g. --SO₃ H) amino acids.

Other substitutions may include unnatural alkylated amino acids which are made by combining an alkyl group with any natural amino acid. Basic natural amino acids such as lysine, arginine may be substituted with alkyl groups at NH₂. Others are nitrile derivatives (e.g., containing the CN-moiety in place of CONH₂) of asparagine or glutamine, and sulfoxide derivative of methionine. In addition, any amide linkage in the peptide can be replaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide, thioamide or reversed amide moieties, e.g. (--C=O)--CH₂ --), (--CHOH)--CH₂ --), (CH₂ --CH₂ --), (--C=S)--NH--), or (--NH--(--C=O) for (--C=O)--NH--).

Compounds of the invention can be prepared, for example, by replacing, deleting, or inserting an amino acid residue of a peptide compound or domain of the invention, with other conservative amino acid residues, i.e., residues having similar physical, biological, or chemical properties, and screening for biological function. It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. The peptides, ligands and domains of the present invention also extend to biologically equivalent peptides, ligands and domains that differ from a portion of the sequence of novel ligands of the present invention by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)s are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.

In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio. 179:125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically, encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include t-butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as --OH, --SH, --CN, --F, --Cl, --Br, --I, --NO₂, --NO, --NH₂, --NHR, --NRR, --C(O)R, --C(O)OH, --C(O)OR, --C(O)NH₂, --C(O)NHR, --C(O)NRR; etc., where R is independently (C₁ -C₆) alkyl, substituted (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, substituted (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, substituted (C₁ -C₆) alkynyl, (C₅ -C₂₀) aryl, substituted (C₅ -C₂₀) aryl, (C₆ -C₂₆) alkaryl, substituted (C₆ -C₂₆) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr, and Tryp, while non-genetically encoded aromatic amino acids include phenylglycine, 2-napthylalanine, beta-2-thienylalanine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine3-fluorophenylalanine, and 4-fluorophenylalanine.

An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held, equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met, while non-genetically encoded apolar amino acids include cyclohexylalanine. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile, while non-genetically encoded aliphatic amino acids include norleucine.

A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln, while non-genetically encoded polar amino acids include citrulline, N-acetyl lysine, and methionine sulfoxide.

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.

The above classifications are not absolute and an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.

Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid. Thus, these substitutions can include compounds whose free amino groups have been derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Similarly, free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides, and side chains can be derivatized to form O-acyl or O-alkyl derivatives for, free hydroxyl groups or N-im-benzylhistidine for the imidazole nitrogen of histidine. Peptide analogues also include amino acids that have been chemically altered, for example, by methylation, by amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, or ethylene diamine, or acylation or methylation of an amino acid side chain (such as acylation of the epsilon amino group of lysine). Peptide analogues can also include replacement of the amide linkage in the peptide with a substituted amide (for example, groups of the formula --C(O)--NR, where R is (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, substituted (C₁ -C₆) alkyl, substituted (C₁ -C₆) alkenyl, or substituted (C₁ -C₆) alkynyl) or isostere of an amide linkage (for example, --CH₂ NH--, --CH₂ S, --CH₂ CH₂ --, --CH=CH-- (cis and trans), --C(O)CH₂ --, --CH(OH)CH₂ --, or --CH₂ SO--).

In order to improve the pharmaceutical characteristics of the R-14 antagonists, the size of the peptides can be reduced by deleting one or more amino acids and use amino acid mimetics or dipeptide mimics containing non-peptide bonds. Examples of using molecular scaffolds such as benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, .beta.-turn dipeptide cores and .beta.-aminoalcohols for these purposes are known to peptide chemists and are described in in Peptidomimetic protocols (Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 & 2 A. Abell (Ed).

Covalent modifications of the peptide are thus included within the scope of the present invention. Such modifications may be introduced into the R-14 antagonists by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation. Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters/e.g. as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosinyl residues per se is well-known, such as for introducing spectral labels into tyrosinyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosinyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'--N=C=N--R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Other modifications of the peptides in the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.

Covalent attachment of fatty acids (C6-C18) to the peptides confer additional biological properties such as protease resistance, plasma protein binding, increased plasma half life, intracellular penetration etc. The above description of modification of a R14 antagonist peptides does not limit the scope of the approaches nor the possible modifications that can be engineered.

Peptides or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA technology using standard methods. Accordingly, the invention further provides nucleic acids that encode peptide compounds of the invention. Such nucleic acids may be introduced into cells for expression using standard recombinant techniques for stable or transient expression. Nucleic acid molecules of the invention may include any chain of two or more nucleotides including naturally occurring or non-naturally occurring nucleotides or nucleotide analogues.

Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject having elevated intraocular pressure and/or glaucoma and related conditions. Therefore, the invention further provides a method for lowering intraocular pressure or for treating a condition associated with elevated intraocular pressure, such as glaucoma and related conditions, in a subject, the method comprising administration of an agent which is capable of R-14 antagonist activity. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the R-14 aberrancy, such that glaucoma is prevented or, alternatively, its progression delayed. In general, the prophylactic or therapeutic methods comprise administering to the subject an effective amount of a compound which is capable of antagonizing a wildtype R-14 activity or agonizing a mutant (defective) R-14 activity. Examples of suitable compounds include the antagonists, agonists or homologues described in detail herein.

Effective Dose

Toxicity and therapeutic efficacy of agents capable of modulating R-14 activity, such as R-14 agonists or antagonists, can be determined by standard pharmaceutical procedures in experimental animals, e.g., for determining The LD₅₀ (The Dose Lethal To 50% Of The Population) and The ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀. Compounds which exhibit large therapeutic induces are preferred. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) determined in in vitro and ex vivo assays and animal studies. Such information can be used to more accurately determine useful doses in humans. Levels of R-14 therapeutics in plasma may be measured, for example, by high performance liquid chromatography. The effective dose of a R-14 therapeutic (agonist or antagonist) could be 0.01 micrograms-100 mg and is determined by the route of administration, pharmaceutical preparation and the mode of delivery.

Formulation and Use

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. Techniques and formulations generally may be found in Reminington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.). For topical administration, R-14 therapeutics of the invention are formulated into solutions, ointments, salves, gels, or creams as generally known in the art. For example, a solution containing a R-14 therapeutic can be applied as drops directly on the eye to lower intraocular pressure.

In one embodiment, such compositions include an agent capable of modulating R-14 activity, such as an R-14 antagonist, in a therapeutically or prophylactically effective amount sufficient to reduce intraocular pressure, and a pharmaceutically acceptable carrier.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the R-14 antagonists can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g. R-14 antagonist) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accordance with an alternative aspect of the invention, an R-14 antagonist may be formulated with one or more additional compounds that enhance the solubility of the R-14 antagonist.

A further form of administration is to the eye. An agent or compound capable of modulating R-14 activity, such as an R-14 antagonist, may be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The pharmaceutically-acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material. Alternatively, the compounds of the invention may be injected directly into the vitreous and aqueous humour. In a further alternative, the compounds may be administered systemically, such as by intravenous infusion or injection, for treatment of the eye.

A further aspect of the present invention is a method of lowering intraocular pressure in a subject and/or preventing and/or treating a condition associated with elevated intraocular pressure (e.g. glaucoma), by administering to a subject a nucleic acid molecule encoding a peptide compound of the invention. Suitable methods of administration include gene therapy methods.

A nucleic acid of the invention may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson el al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel el al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Defective retroviruses, are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76:271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

For use as a gene therapy vector, the genome of an adenovirus may be manipulated so that it encodes and expresses a peptide compound of the invention, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin el al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).

Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790). Lentiviral gene therapy vectors may also be adapted for use in the invention.

General methods for gene therapy are known in the art. See for example, U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and Cavazzana-Calvo et al., Science 288:669-72 (2000)).

The invention further relates to transplantation methods, to introduce into a subject a cell comprising a nucleic acid capable of encoding a peptide compound of the invention. The nucleic acid may be present in a vector as described above, the vector being introduced into the cell in vitro, using for example the methods described above. In an embodiment, the cell is autologous, and is obtained from the subject. In embodiments, the cell is allogeneic or xenogeneic.

In embodiments, the therapeutic method may be used in conjunction with a diagnostic method. For example, a subject suffering from a condition associated with intraocular pressure (e.g. glaucoma) may be identified or diagnosed using a diagnostic method and then subsequently treated using a therapeutic method. Further, the therapeutic method may be used for treatment in conjunction with the diagnostic or prognostic method which is used to monitor the progress of the treatment.

In accordance with another aspect of the invention, therapeutic compositions of the present invention, comprising a R-14 antagonist, may be provided in containers or commercial packages which further comprise instructions for use of the R-14 antagonist for the prevention and/or treatment of elevated intraocular pressure and related disorders such as glaucoma.

Accordingly, the invention further provides a commercial package comprising an R-14 antagonist or the above-mentioned composition together with instructions for the prevention and/or treatment of elevated intraocular pressure and related disorders such as glaucoma.

The invention further provides a use of the above-noted peptides, compounds and compositions for lowering intraocular pressure in a subject and/or for the prevention and/or treatment of elevated intraocular pressure and related disorders such as glaucoma.

The invention further provides a use of the above-noted peptides, compounds and compositions for the preparation of a medicament for lowering intraocular pressure in a subject and/or for the prevention and/or treatment of elevated intraocular pressure and related disorders such as glaucoma.

Claim 1 of 29 Claims

What is claimed is:

1. A substantially pure peptide compound of Formula I:

Z₁ -Ile-Phe-Asp-Ser-Thr-Glu-Cys-Trp-Z₂ I

wherein:

Z₁ is an N-terminal group of the formula H₂ N--, RHN-- or, RRN--;

Z₂ is a C-terminal group of the formula --C(O)OH, --C(O)R, --C(O)OR, --C(O)NHR, --C(O)NRR;

R at each occurrence is independently selected from (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, substituted (C₁ -C₆) alkyl, substituted (C₁ -C₆) alkenyl, or substituted (C₁ -C₆) alkynyl; and "--" is a covalent linkage.

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