Patent Application
20080089881
This invention is based on the discovery that some GPCR genes, HM74 and HM74A are up-regulated in inflammatory disease tissue cells. Accordingly, the invention provides methods for diagnosing and treating inflammatory diseases by targeting these GPCR genes.
A diagnostic method of the invention involves comparing the gene expression or protein activity level of HM74 and/or HM74A in a sample prepared from a subject with that in a sample prepared from a normal subject, i.e., a subject who does not suffer from inflammatory diseases. A higher gene expression or protein activity level of HM74 and/or HM74A indicates that the subject is suffering from or at risk for developing inflammatory diseases. For example, if the gene expression level in a test subject is significant different than that in a normal subject as determined by the method described in the examples below or any analogous methods, the test subject is identified as being suffering from or at risk for developing inflammatory diseases. The method of the invention can be used on its own or in conjunction with other procedures to diagnose inflammatory diseases.
The gene expression level of HM74 and/or HM74A can be determined at either the mRNA level or the protein level. Methods of measuring mRNA levels in a tissue sample are known in the art. In order to measure mRNA levels, cells can be lysed and the levels of mRNA in the lysates or in RNA purified or semi-purified from the lysates can be determined by any of a variety of methods including, without limitation, hybridization assays using detectably labeled gene-specific DNA or RNA probes and quantitative or semi-quantitative RT-PCR or TaqMan real time PCR methodologies using appropriate gene-specific oligonucleotide primers. Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out using, for example, tissue sections or unlysed cell suspensions, and detectably (e.g., fluorescently or enzyme) labeled DNA or RNA probes. Additional methods for quantifying mRNA include RNA protection assay (RPA) and SAGE.
Methods of measuring protein levels in a tissue sample are also known in the art. Many such methods employ antibodies (e.g., monoclonal or polyclonal antibodies) that bind specifically to the target protein. In such assays, the antibody itself or a secondary antibody that binds to it can be detectably labeled. Alternatively, the antibody can be conjugated with biotin, and detectably labeled avidin (a polypeptide that binds to biotin) can be used to detect the presence of the biotinylated antibody. Combinations of these approaches (including “multi-layer sandwich” assays) familiar to those in the art can be used to enhance the sensitivity of the methodologies. Some of these protein-measuring assays (e.g., ELISA or Western blot) can be applied to lysates of cells, and others (e.g., immunohistological methods or fluorescence flow cytometry) applied to histological sections or unlysed cell suspensions. Methods of measuring the amount of label depend on the nature of the label and are well known in the art. Appropriate labels include, without limitation, radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S, .sup.3H, or .sup.32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or .beta.-glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot.™. nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Other applicable assays include quantitative immunoprecipitation or complement fixation assays.
The protein activity level of HM74 and/or HM74A can be determined, e.g., by measuring the binding of nicotinic acid, or by measuring GDP-GTP exchange on G-protein subunits following ligand-induced activation of HM74/HM74A See, Peltonen et al. (1998) Eur J Pharmacol 355, 275; Tunaru et al (2003) Nature Medicine 9, 352-355; Wise et al, (2003) J Biol Chem 278, 9869-9874.
The invention also provides a method for identifying and manufacturing molecules (such as, polynucleotides, RNA interference agent, an anti-sense RNA, siRNA, proteins, peptides, peptidomimetics, peptoids, antibodies or their variants, or non-peptidyl small molecules) that decrease the gene expression or protein activity level of HM74 and/or HM74A in a system. Molecules thus identified can be used, e.g., for treating inflammatory diseases.
The candidate molecules can be obtained using any of the numerous approaches in combinatorial library methods known in the art. Such libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the “one-bead one-compound” libraries. See, e.g., Zuckermann et al. (1994) J Med Chem 37, 2678-2685; and Lam (1997) Antiinflammatory diseases Drug Des 12, 145.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al. (1993) PNAS USA 90, 6909; Erb et al. (1994) PNAS USA 91, 11422; Zuckermann et al. (1994) J Med Chem 37, 2678; Cho et al. (1993) Science 261, 1303; Carrell et al. (1994) Angew Chem Int Ed Engl 33, 2059; Carell et al. (1994) Angew Chem Int Ed Engl 33, 2061; and Gallop et al. (1994) J Med Chem 37, 1233. Methods of making monoclonal and polyclonal antibodies and fragments thereof are also known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term “antibody” includes intact molecules and fragments thereof, such as Fab, F(ab′).sub.2, and Fv which are capable of binding to an epitopic determinant present in the HM74 and/or HM74A protein.
Libraries of molecules may be presented in solution (e.g., Houghten (1992) Biotechniques 13, 412-421), or on beads (Lam (1991) Nature 354, 82-84), chips (Fodor (1993) Nature 364, 555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) PNAS USA 89, 1865-1869), or phages (Scott and Smith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406; Cwirla et al. (1990) PNAS USA 87, 6378-6382; Felici (1991) J Mol Biol 222, 301-310; and U.S. Pat. No. 5,223,409).
To identify molecules that decrease the gene expression or protein activity level of HM74 and/or HM74A in a subject, a system containing the HM74 and/or HM74A gene or an HM74 and/or HM74A gene product (mRNA or protein) is contacted with a candidate compound, and the gene expression or protein activity level of HM74 and/or HM74A is evaluated relative to that in the absence of the candidate compound. In a cell system, the cell (e.g., a inflammatory diseases cell) can be a cell that naturally expresses the HM74 and/or HM74A gene, or a cell that is modified to express a recombinant HM74 and/or HM74A gene, for example, by having the HM74 and/or HM74A gene fused to a heterologous promoter or by having the HM74 and/or HM74A promoter fused to a heterologous gene. The gene expression or protein activity level of HM74 and/or HM74A can be determined according to the methods described in the examples below, or any other methods well known in the art. If the gene expression or protein activity level of HM74 and/or HM74A is lower in the presence of the candidate molecule than that in the absence of the candidate compound, the candidate molecule is identified as being useful for treating inflammatory diseases.
This invention further provides a method for treating inflammatory diseases. Subjects to be treated can optionally be identified, for example, by determining the gene expression or protein activity level of HM74 and/or HM74A in a sample prepared from a subject by methods described above. If the gene expression or protein activity level of HM74 and/or HM74A is higher in the sample from the subject than that in a sample from a normal subject, the subject is a candidate for treatment with an effective amount of a compound that decreases the gene expression or protein activity level of HM74 and/or HM74A in the subject. This method can be performed alone or in conjunction with other drugs or therapy.
The term “treating” is defined as administration of a composition to a subject, who has inflammatory diseases, with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder. An “effective amount” is an amount of the composition that is capable of producing a medically desirable result, e.g., as described above, in a treated subject.
In one in vivo approach, a therapeutic composition (e.g., a composition containing a compound identified as described above) is administered to the subject. Generally, the molecule is suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. For treatment of inflammatory diseases, the compound can be delivered directly to the inflammatory diseases tissue.
The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery. If the compound is poorly soluble, known surfactant materials may be used to enhance solubility of the compound. See, for example, Li et al., U.S. Application Publication No. 2006/0068007 A1, incorporated herein by reference and describing a modified Vitamin E TPGS as a surfactant material.
Alternatively, a polynucleotide, such as one containing a nucleic acid sequence encoding an anti-sense HM74 and/or HM74A RNA, can be delivered to the subject, for example, by the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art. Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. (1995) J Mol Med 73, 479). Alternatively, tissue specific targeting can be achieved by the use of tissue-specific transcriptional regulatory elements (TRE) which are known in the art. Delivery of “naked DNA” (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
The above-described polynucleotide can be an RNA interference agent, i.e., a duplex-containing RNA or a DNA sequence encoding it, which inhibits the expression of HM74 and/or HM74A via RNA interference. RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) directs homologous sequence-specific degradation of messenger RNA. In mammalian cells, RNAi can be triggered by 19 to 21-nucleotide duplexes of small interfering RNA (siRNA) without activating the host interferon response. As RNAi represses the expression of a specific gene, it can be used to treat a disease caused by abnormally high levels of expression of the gene. A duplex-containing RNA can be synthesized by techniques well known in the art. See, e.g., Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio. 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. It can also be transcribed from an expression vector and isolated using standard techniques.
In the above-mentioned polynucleotides (e.g., expression vectors), the nucleic acid sequence encoding an RNAi agent or an anti-sense HM74 and/or HM74A RNA is operatively linked to a promoter or enhancer-promoter combination. Enhancers provide expression specificity in terms of time, location, and level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription initiation site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site.
Suitable expression vectors include plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.
Polynucleotides can be administered in a pharmaceutically acceptable carrier. As is well known in the medical art, the dosage for any one subject depends upon many factors, including the subject's weight, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for administration of polynucleotide is about 106 to 1012 copies of the polynucleotide molecule. This dose can be repeatedly administered as needed. Routes of administration can be any of those listed above.
Also within the scope of the invention is a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of a compound that decreases the gene expression or protein activity level of HM74 and/or HM74A in a subject. The pharmaceutical composition can be used to treat inflammatory diseases. The pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an isotonic and absorption delaying agent. The molecule can also be packaged in a container with a label or an insert to indicate the intended uses of the compound, i.e., treatment of inflammatory diseases.
The molecule of the invention can be formulated into dosage forms for different administration routes utilizing conventional methods. For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the ligand with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite.
The molecule can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent. The pharmaceutical composition can be administered via the parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Cyclodextrins, or other solubilizing agents well known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.
The efficacy of a composition of the invention can be evaluated both in vitro and in vivo. For example, the composition can be tested for its ability to decrease the level of HM74 and/or HM74A gene expression or protein activity in vitro. For in vivo studies, the composition can be injected into an animal (e.g., an animal model) and its effects on inflammatory diseases are then accessed. Based on the results, an appropriate dosage range and administration route can be determined.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.
We applied Affymetrix gene chips technologies (human U133 chips) to perform gene expression profile studies of the human colon tissues from ulcerative colitis patients and normal donors. We observed that HM74 is highly induced in inflamed colon tissues from ulcerative colitis patients. Further statistical analysis of colon tissues from inflamed bowel disease (IBD) patients and normal donors (9 Crohn's disease patients, 17 Ulcerative colitis patients, 211 normals) revealed that HM74 is induced 4.4 fold (p value, 0.002) in Crohn's disease colons and induced 2.5 fold (p value, 0.006) in Ulcerative colitis colons. The oligonucleotide probes (probe ID 205220_at) on Affymetrix chips detect both the human HM74 and HM74A. In order to identify which HM74 isoform is induced in IBD, we performed TaqMan real time RT-PCR analysis with specific TaqMan primers and probes designed for HM74 or HM74A. As shown in
Total RNA isolation: Total cellular RNA was isolated from tissue or cell samples using the RNeasy Kits and RNase-Free DNase Set Protocol according to manufacturer's description (QIAGEN).
TaqMan probes and primers: PCR primers and TaqMan probes were designed using Primer Express 1.5 Software (Applied Biosystems). The TaqMan probes were labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein), at the 5′ end and a fluorescent dye quencher TAMRA (6-carboxy-tetramethyl-rhodamine) at the 3′ end. The specificity of PCR primers was tested under normal PCR conditions in a thermal cycler prior to TaqMan PCR quantitation.
Reverse transcription: RT reactions were carried out for each RNA sample in MicroAmp reaction tubes using TaqMan reverse transcription reagents. Each reaction tube contained 500 ng of total RNA in a volume of 50 μl containing 1× TaqMan RT buffer, 5.5 mM MgCl2, 500 μM of each dNTP, 2.5 μM of Random Hexamers or oligo-d(T)16 primers, 0.4 U/μl of RNase inhibitor, and 1.25 U/μl of MultiScribe Reverse Transcriptase. RT reactions were carried out at 25° C. for 10 min, 48° C. for 40 min and 95° C. for 5 min [Note: the incubation at 25° C. for 10 min is necessary for the RT reaction with random hexamers or oligo-d(T)16 primers to obtain the optimal results]. The RT reaction mixture was then placed at 4° C. for immediate use of PCR amplification, or stored at −20° C. for later use (similar results are expected at these two different temperatures of storage).
Construction of standard curve: To determine the copy numbers of the target transcript, a human genomic DNA (Clontech) was used to generate a standard curve. The copy numbers of genomic DNA template were calculated according to the molecular weight of human diploid genome [3×109 bp=3×109×660 (M.W.)=2×1012 g], and then 1 μg/μl genomic DNA was converted into 2.4×106 copy numbers based upon the Avogadro's number (1 mol=6.022×1023 molecules). The genomic DNA was serially (every ten-fold) diluted at a range of 5×105 to 5×100 copy numbers. Each sample was run in triplicates, and the Rn (the ratio of the amount of reporter dye emission to the quenching dye emission) and threshold cycle (Ct) values were averaged from each reaction.
TaqMan real-time quantitative PCR: The principle of the TaqMan real-time detection is based on the fluorogenic 5′ nuclease assay. A thermal stable AmpliTaq Gold DNA polymerase was used for the PCR amplification. Real-time RT-PCR was performed in a MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems). Each well contained 2 μl of each RT product (20 ng total RNA), 1× TaqMan buffer A, 5.5 mM MgCl2, 200 μM dATP/dCTP/dGTP, 400 μM dUTP, 200 nM primers (forward and reverse), 100 nM TaqMan probe, 0.01 U/μl AmpErase, and 0.025 U/μl AmpliTaq Gold DNA polymerase in a total volume of 25 μl. Each well was closed with MicroAmp Optical caps (Applied Biosystems), following complete loading of reagents. Amplification conditions were 2 min at 50° C. (for AmpErase UNG incubation to remove any uracil incorporated into the cDNA), 10 min at 95° C. (for AmpliTaq Gold activation), and then run for 40 cycles at 95° C. for 15 s, 60° C. for 1 min. All reactions were performed in the ABI Prism 7700 Sequence Detection System for the test samples, standards, and no template controls. They were run in triplicates using the Sequence Detector V 1.6 program. The Rn and Ct were averaged from the values obtained in each reaction. A “standard curve” was constructed by plotting the Ct vs. the known copy numbers of the template in the standard. According to the standard curve, the copy numbers for all unknown samples were obtained automatically.
Normalization of mRNA expression level: The copy numbers of mRNA in each sample were calculated based on its Ct value with its plasmid DNA standard curve. The copy numbers were then normalized to Gapdh to minimize variability in the results due to differences in the RT efficiency and RNA integrity among test samples.
Primer sequences used for Quantitative RT-PCR
We observed that HM74 is highly induced in inflamed colon tissues from ulcerative colitis patients. Further statistical analysis of colon tissues from inflamed bowel disease (IBD) patients and normal donors (9 Crohn's disease patients, 17 Ulcerative colitis patients, 211 normals) revealed that HM74 is induced 4.4 fold (p value, 0.002) in Crohn's disease colons and induced 2.5 fold (p value, 0.006) in Ulcerative colitis colons. As shown in
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.
This application claims benefit from U.S. Provisional Application No. 60/748,313, filed on Dec. 7, 2005.
| Number | Date | Country | |
|---|---|---|---|
| 60748313 | Dec 2005 | US |
