Title: Method of diagnosing ischemic stroke via UCP-2 detection
United States Patent: 6,670,138
Issued: December 30, 2003
Inventors: Gonzalez-Zulueta; Mirella (Pacifica, CA); Shamloo; Mehrdad (San Mateo, CA); McFarland; K.C. (Davis, CA); Chin; Daniel (Foster City, CA); Wieloch; Tadeusz (Lund, SE); Melcher; Thorsten (San Francisco, CA)
Assignee: AGY Therapeutics, Inc. (South San Francisco, CA)
Appl. No.: 001051
Filed: October 31, 2001
The present invention identifies a gene whose gene product provides a protective effect against neurological disorders or neuronal injuries. Further, the invention provides methods for diagnosing or assessing an individual's susceptibility to a neuronal injury such as stroke. Also provided are therapeutic methods for treating patients, and methods for prophylactically treating individuals susceptible to various neurological disorders or neuronal injuries. Additionally, the invention describes screening methods for identifying agents that can be administered to treat individuals that have suffered or are at risk to suffer such disorders or injuries.
DETAILED DESCRIPTION OF THE INVENTION
Overview of the invention
A variety of methods for diagnosing and treating individuals that have either suffered a neurological injury, that are at risk for neurological injury, or that have a neurological disorder are provided. High throughput screening methods to identify compounds effective in treating such individuals are also provided, as are compositions that include compounds identified through such screening methods. The methods and compositions find utility with a variety of neurological disorders, including stroke and, more specifically, ischemic stroke. The methods are based in part upon the finding that UCP-2 is differentially expressed (upregulated) in an ischemic preconditioning model in rat, indicating that UCP-2 exerts a protective effect against various neurological disorders, particularly stroke.
Certain methods are also based upon evidence indicating that UCP-2 inhibits certain components of an apoptotic cascade (see, e.g., Example 4). More specifically, the evidence indicates that UCP-2 may interfere with the effects of mitochondrial Ca2+ accumulation and the subsequent mitochondrial permeability transition (see, e.g., Example 4). Mitochondria have a large capacity for buffering Ca2+, and during various toxic stimuli mitochondria accumulate large quantities of Ca2+. However, excessive mitochondrial Ca2+ overload interfere with mitochondrial ATP production and lead to opening of the permeability transition pore (PTP). The PTP is a voltage-sensitive proteinaceous pore that allows solutes of <1,500 Daltons to equilibrate across the membrane (see D. G. Nicholls and S. L. Budd (2000) Physiological Reviews 80: 315-361). Opening of the pore results in dissipation of the mitochondrial membrane potential and mitochondrial swelling. Mitochondrial swelling induces release of cytochrome c into the cytosol where it interacts with apoptotic mediators. Pore formation can be inhibited, amongst other factors, by low matrix pH. UCP-2 is a mitochondrial proton transporter that leaks protons into the mitochondrial matrix. Thus, while not intending to be bound by any particular theory, it may be that activation of UCP-2 leads to a decreased matrix pH and subsequent prevention of PTP opening.
In view of the increase in UCP-2 expression observed in response to a neurological insult, the diagnostic and prognostic methods generally involve detecting the occurrence of a stroke or assessing an individual's susceptibility to stroke by detecting an elevated level of UCP-2 expression or activity in a sample obtained from the patient. Because of the protective effect provided by UCP-2, both therapeutic and prophylactic treatment methods for individuals suffering or at risk of a neurological disorder such as stroke involve administering either a therapeutic or prophylactic amount of an agent that increases the activity of UCP-2. The agent that acts to increase UCP-2 activity can be a purified form of UCP-2, an agent that stimulates expression or synthesis of UCP-2, or a nucleic acid that includes a segment encoding UCP-2, or any agent that acts as an activator of the UCP-2 activity and function including but not limited to pharmacological agonists, or partial agonists. In view of the role of UCP-2 role as a potential regulator of mitochondrial permeability transition and release of cytochrome c, as well as an inhibitor of caspase-3 activation, the agent can also be one that has similar effects on PTP, cytochrome c release and caspase-3 activation.
The screening methods generally involve conducting various types of assays to identify agents that upregulate the expression or activity of UCP-2. Such screening methods can initially involve screens to identify compounds that can bind to UCP-2. Certain assays are designed to measure more clearly the effect that different agents have on UCP-2 activities or expression levels. Other screening methods are designed to identify compounds that influence mitochondrial permeability transition and inhibit caspase-3 activation as does UCP-2. Lead compounds identified during these screens can serve as the basis for the synthesis of more active analogs. Lead compounds and/or active analogs generated therefrom can be formulated into pharmaceutical compositions effective in treating neurological disorders such as stroke.
III. Differential Expression of UCP-2
The mammalian brain has a limited capacity to survive long periods of hypoxia and ischemia (lack of oxygen and blood supply). Following exposure to hypoxia-ischemia, neurons die by rapid or slow mechanisms of cell death (necrosis or apoptosis). Hypoxic-ischemic brain insults, such as stroke, neonatal asphyxia, heart failure (prolonged lack of blood supply to the brain), or drowning, can cause severe permanent brain damage.
On the other hand, brief, sublethal periods of hypoxia-ischemia can lead to a transient phase in the brain when neurons become protected from subsequent injury and death. This treatment, generally referred to as ischemic tolerance, or ischemic preconditioning, can provide the basis for and lead to an understanding of intrinsic protective mechanisms and pathways through endogenous proteins or factors that provide for this effect. Thus, as used herein, ischemic preconditioning refers to a brief, transient, non-destructive stroke that triggers intrinsic neuroprotective mechanisms.
Stroke can be modeled in animals, such as the rat, by occluding certain cerebral arteries that prevent blood from flowing into particular regions of the brain, then releasing the occlusion and permitting blood to flow back into that region of the brain (reperfusion). These focal ischemia models are different than global ischemia models where blood flow to the entire brain is blocked for a period of time prior to reperfilsion. Certain regions of the brain are particularly sensitive to this type of global ischemic insult. The hippocampus, and more specifically the CA1 region of the hippocampus, is primarily affected by global ischemia. Ten minutes of global ischemia induce profound selective neuronal loss in the CA1 region of hippocampus with non-detectable neuronal damage in CA3 region and dentate gyrus of hippocampus. With increasing periods of global ischemia, delayed cell death can also be detected in the striatum and layers 2 and 5 of the cerebral cortex (Lipton (1999) Physiol. Rev. 79: 1431-1568). In contrast, in focal ischemia, the precise region that is directly damaged is dictated by the location of the blockade and duration of ischemia prior to reperfusion. In animal models of focal ischemia there is, like in the human condition, a gradation of ischemia from the infarct core of the lesion to the outermost boundary, and hence there are different metabolic conditions within the affected site. Because of its duration and heterogeneity, the insult is complex.
In the rat, ten minutes global ischemia (Two-Vessel occlusion model with hypotension, Lipton (1999) Physiol. Rev. 79: 1431-1568) is sufficient to induce the complete destruction of CA1 neurons in the hippocampus. However, a three-minute ischemic event and several hours of recovery time are sufficient to effectively reduce the damage of a ten-minute ischemic insult. This neuroprotective effect is dependent on de novo protein synthesis. Therefore, genes that are specifically upregulated in an ischemic preconditioning model may be neuroprotective, either directly or indirectly, whereas longer ischemic times may lead to the induction of other genes that have neuro-damaging properties. The rat model of both ischemic preconditioning and global ischemia is highly relevant because it duplicates the ischemia/reperfusion that occurs in the human brain during drowning, cardiac by-pass surgery and cardiac arrest.
As described in greater detail in Example 1, a number of genes that are induced in the hippocampus by such protective hypoxic-ischemic treatment have been identified using rat model systems. These genes were identified by performing differential cloning between preconditioned and normal rat brains and sequencing the differentially expressed genes. This sequence information was subsequently utilized to conduct sequence comparisons with sequences available in public databases using standard sequence algorithms (e.g., BLAST). Of the differentially expressed genes identified, four independent clones were identified that match the sequence for rat UCP-2 (Genbank ID: AB010743). UCP-2 upregulation in an ischemic preconditioning model in which preconditioning confers a protective effect against subsequent neurological insults indicates that an increase in UCP-2 activity can have a neuroprotective effect on various neurological cell types (e.g., neurons, glial cells, microglial cells), thereby protecting against various neurological disorders, including but not limited to, stroke and ischemic stroke. Thus, agents able to increase the expression levels or activity of UCP-2 can potentially have a neuroprotective effect.
The role that UCP-2 plays in regulating mitochondrial membrane potential also indicates that agents that alter mitochondrial permeability are also candidates for providing a neuroprotective effect. As described more fully in the Examples below, the evidence indicates that the mechanism of action for UCP-2 involves inhibition of cell apoptosis, which in turn is a consequence of a cascade of events involving the mitochondrial permeability transition.
Apoptosis, or programmed cell death, plays a fundamental role in normal biological processes as well as in several disease states (see, e.g., Nicholson and Thornberry, (1996) Trends Biochem. Sci. 22:299-306; and Thompson (1995) Science 281:1312-1316). Apoptosis can be induced by various stimuli that all produce the same end result: systematic and deliberate cell death. One apoptotic cascade is triggered by mitochondrial permeability transition which consists in the opening of a voltage-sensitive pore that allows solutes to equilibrate across the mitochondrial membrane. Mitochondria participate in apoptotic signaling by mediating the activation of caspases via release of cytochrome c to the cytosol. Thus, localization of cytochrome c serves as a convenient marker for studying mitochondrial involvement in apoptosis. Caspases are cysteine proteases that possess the unusual ability to cleave substrates after aspartate residues; this activity is central to their role in apoptosis. Upon activation, caspases disable cellular homeostatic and repair processes, and cleave important structural components in the cell. Caspase-3 plays a direct role in proteolytic digestion of cellular proteins responsible for progression to apoptosis (see, e.g., Fernandes-Alnemri et al. (1994) J. Biol. Chem. 269:30761-30764).
The mechanisms underlying the neuroprotective role of UCP-2 may include inhibition and/or regulation of any of the components of the apoptotic cascade including, but not limited to, effects on mitochondrial membrane potential and mitochondrial permeability transition, blockade of cytochrome c release from mitochondria, and activation of caspases, as evidenced in the Examples below. That UCP-2 is a mitochondrial protein capable of lowering the mitochondrial matrix pH, reducing free-radical levels and ATP production, which are involved in neuronal apoptosis, is consistent with such a mechanism.
The finding of UCP-2 upregulation as a mechanism for providing a protective neurological effect and the evidence indicating its role in inhibiting apoptosis provides the basis for a number of diagnostic and therapeutic methods, as well as screening methods. Agents that influence UCP-2 activity or expression can potentially provide a more effective neuroprotective effect than agents that interact with a downstream component of an apoptotic cascade (e.g., caspase 3). Because UCP-2 occurs early in the apoptotic cascade, it has the potential to affect a greater number of cellular pathways than a component that is further downstream in the cascade. These various diagnostic, treatment and screening methods are discussed further below.
IV. Diagnostic and Prognostic Methods
The differential expression of UCP-2 in response to an ischemic event indicates that UCP-2 can serve as a marker for diagnosing individuals that have suffered a mild stroke, and in prognostic evaluations to detect individuals at risk for stroke. Prognostic methods can also be utilized in the assessment of the severity of the stroke and the likelihood of recovery.
In general, such diagnostic and prognostic methods involve detecting an elevated level of UCP-2 in the cells or tissue of an individual or a sample therefrom. A variety of different assays can be utilized to detect an increase in UCP-2 expression, including both methods that detect UCP-2 transcript and UCP-2 protein levels. More specifically, the diagnostic and prognostic methods disclosed herein involve obtaining a sample from an individual and determining at least qualitatively, and preferably quantitatively, the level of UCP-2 expression in the sample. Usually this determined value or test value is compared against some type of reference or baseline value. Details regarding samples, methods for quantitating expression levels and controls are set forth in the following sections.
A. Detection of Transcript
A number of different methods for detecting and optionally quantitating UCP-2 transcript are available and known to those of skill in the art. Examples of suitable methods for detecting an quantifying changes in UCP-2 expression include, but are not limited to, dot blots, in-situ hybridization, nucleic acid probe arrays, quantitative reverse-transcription PCR, (RT-PCR), Northern blots and RNAase protection methods.
1. Dot Blots and In-situ Hybridization
Dot blots are one example of an assay that can be utilized to determine the amount of UCP-2 transcript present in a nucleic acid sample obtained from an individual being tested. In these assays, a sample from an individual being tested for stroke is spotted on a support (e.g., a filter) and then probed with labeled nucleic acid probes that specifically hybridize with UCP-2 nucleic acids. After the probes have been allowed to hybridize to the immobilized nucleic acids on the filter, unbound nucleic acids are rinsed away and the presence of hybridization complexes detected and quantitated on the basis of the amount of labeled probe bound to the filter.
In-situ hybridization methods are hybridization methods in which the cells are not lysed prior to hybridization. Because the method is performed in situ, it has the advantage that it is not necessary to prepare RNA from the cells. The method usually involves initially fixing test cells to a support (e.g., the walls of a microtiter well) and then permeabilizing the cells with an appropriate permeabilizing solution. A solution containing labeled probes for UCP-2 is then contacted with the cells and the probes allowed to hybridize with UCP-2 nucleic acids. Excess probe is digested, washed away and the amount of hybridized probe measured. This approach is described in greater detail by Harris, D. W. (1996) Anal. Biochem. 243:249-256; Singer, et al. (1986) Biotechniques 4:230-250; Haase et al. (1984) Methods in Virology, vol. VII, pp. 189-226; and Nucleic Acid Hybridization: A Practical Approach (Hames, et al., eds., 1987).
The hybridization probes utilized in the foregoing methods are polynucleotides that are of sufficient length to specifically hybridize to a UCP-2 nucleic acid. Hybridization probes are typically at least 15 nucleotides in length, in some instances 20 to 30 nucleotides in length, in other instances 30 to 50 nucleotides in length, and in still other instances up to the full length of a UCP-2 nucleic acid. The probes are labeled with a detectable label, such as a radiolabel, fluorophore, chromophore or enzyme to facilitate detection. Methods for synthesizing the necessary probes include the phosphotriester method described by Narang et al. (1979) Methods of Enzymology 68:90, and the phosphodiester method disclosed by Brown et al. (1979) Methods of Enzymology 68:109.
2. Nucleic Acid Probe Arrays
Related hybridization methods utilize nucleic acid probe arrays to detect and quantitate UCP-2 transcript. The arrays utilized to detect UCP-2 can be of varying types. The probes utilized in the arrays can be of varying types and can include, for example, synthesized probes of relatively short length (e.g., a 20-mer or a 25-mer), cDNA (full length or fragments of gene), amplified DNA, fragments of DNA (generated by restriction enzymes, for example) and reverse transcribed DNA (see, e.g., Southern et al. (1999) Nature Genetics Supplement 21:5-9 (1999). Both custom and generic arrays can be utilized in detecting UCP-2 expression levels. Custom arrays can be prepared using probes that hybridize to particular preselected subsequences of mRNA gene sequences of UCP-2 or amplification products prepared from them. Generic arrays are not specially prepared to bind to UCP-2 sequences but instead are designed to analyze mRNAs irrespective of sequence. Nonetheless, such arrays can still be utilized because UCP-2 nucleic acids only hybridize to those locations that include complementary probes. Thus, UCP-2 levels can still be determined based upon the extent of binding at those locations bearing probes of complementary sequence.
In probe array methods, once nucleic acids have been obtained from a test sample, they typically are reversed transcribed into labeled cDNA, although labeled mRNA can be used directly. The test sample containing the labeled nucleic acids is then contacted with the probes of the array. After allowing a period sufficient for any labeled UCP-2 nucleic acid present in the sample to hybridize to the probes, the array is typically subjected to one or more high stringency washes to remove unbound nucleic acids and to minimize nonspecific binding to the nucleic acid probes of the arrays. Binding of labeled UCP-2 is detected using any of a variety of commercially available scanners and accompanying software programs.
For example, if the nucleic acids from the sample are labeled with fluorescent labels, hybridization intensity can be determined by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by e.g., U.S. Pat. No. 5,578,832 to Trulson et al., and U.S. Pat. No. 5,631,734 to Stem et al. and are available from Affymetrix, Inc., under the GeneChip.TM. label. Some types of label provide a signal that can be amplified by enzymatic methods (see Broude, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 3072-3076 (1994)). A variety of other labels are also suitable including, for example, radioisotopes, chromophores, magnetic particles and electron dense particles.
Those locations on the probe array that are hybridized to labeled nucleic acid are detected using a reader, such as described by U.S. Pat. No. 5,143,854, WO 90/15070, and U.S. Pat. No. 5,578,832. For customized arrays, the hybridization pattern can then be analyzed to determine the presence and/or relative amounts or absolute amounts of known mRNA species in samples being analyzed as described in e.g., WO 97/10365.
Further guidance regarding the use of probe arrays sufficient to guide one of skill in the art is provided in WO 97/10365, PCT/US/96/143839 and WO 97/27317. Additional discussion regarding the use of microarrays in expression analysis can be found, for example, in Duggan, et al., Nature Genetics Supplement 21:10-14 (1999); Bowtell, Nature Genetics Supplement 21:25-32 (1999); Brown and Botstein, Nature Genetics Supplement 21:33-37 (1999); Cole et al., Nature Genetics Supplement 21:38-41 (1999); Debouck and Goodfellow, Nature Genetics Supplement 21:48-50 (1999); Bassett, Jr., et al., Nature Genetics Supplement 21:51-55 (1999); and Chakravarti, Nature Genetics Supplement 21:56-60 (1999).
3. Quantitative RT-PCR
A variety of so-called "real time amplification" methods or "real time quantitative PCR" methods can also be utilized to determine the quantity of UCP-2 mRNA present in a sample. Such methods involve measuring the amount of amplification product formed during an amplification process. Fluorogenic nuclease assays are one specific example of a real time quantitation method that can be used to detect and quantitate UCP-2 transcript. In general such assays continuously measure PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe--an approach frequently referred to in the literature simply as the "TaqMan" method.
The probe used in such assays is typically a short (ca. 20-25 bases) polynucleotide that is labeled with two different fluorescent dyes. The 5' terminus of the probe is typically attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes can be attached at other locations on the probe as well. For measuring UCP-2 transcript, the probe is designed to have at least substantial sequence complementarity with a probe binding site on UCP-2 transcript. Upstream and downstream PCR primers that bind to regions that flank UCP-2 are also added to the reaction mixture for use in amplifying UCP-2.
When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter dye from the polynucleotide-quencher complex and resulting in an increase of reporter emission intensity that can be measured by an appropriate detection system.
One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 manufactured by Applied Biosystems, Inc. in Foster City, Calif. Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. These recorded values can then be used to calculate the increase in normalized reporter emission intensity on a continuous basis and ultimately quantify the amount of the mRNA being amplified.
Additional details regarding the theory and operation of fluorogenic methods for making real time determinations of the concentration of amplification products are described, for example, in U.S. Pat. No. 5,210,015 to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No. 5,863,736 to Haaland, as well as Heid, C. A., et al., Genome Research, 6:986-994 (1996); Gibson, U. E. M, et al., Genome Research 6:995-1001 (1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K. J., et al., PCR Methods and Applications 357-362 (1995), each of which is incorporated by reference in its entirety.
4. Northern Blots
Northern blots can be used to detect and quantitate UCP-2 transcript. Such methods typically involve initially isolating total cellular or poly(A) RNA and separating the RNA on an agarose gel by electrophoresis. The gel is then overlaid with a sheet of nitrocellulose, activated cellulose, or glass or nylon membranes and the separated RNA transferred to the sheet or membrane by passing buffer through the gel and onto the sheet or membrane. The presence and amount of UCP-2 transcript present on the sheet or membrane can then be determined by probing with a labeled probe complementary to UCP-2 to form labeled hybridization complexes that can be detected and optionally quantitated (see, e.g., . Sambrook, et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed) Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY).
5. RNAase Protection Assays
Ribonuclease protection assays (RPA) involve preparing a labeled antisense RNA probe for UCP-2. This probe is subsequently allowed to hybridize in solution with UCP-2 transcript contained in a biological sample to form RNA:RNA hybrids. Unhybridized RNA is then removed by digestion with an RNAase, while the RNA:RNA hybrid is protected from degradation. The labeled RNA:RNA hybrid is separated by gel electrophoresis and the band corresponding to UCP-2 detected and quantitated. Usually the labeled RNA probe is radiolabeled and the UCP-2 band detected and quantitated by autoradiography. RPA is discussed further by (Lynn et al. (1983) Proc. Natl. Acad. Sci. 80:2656; Zinn, et al. (1983) Cell 34:865; and Sambrook, et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed) Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY).
B. Detection of UCP-2 Translation Product
Instead of detecting an increase in transcript, another option for detecting UCP-2 expression is to determine UCP-2 protein levels and/or activity. A number of different approaches can be utilized to accomplish this, including the use of antibodies that specifically bind UCP-2 and assays that measure UCP-2 activity (e.g., mitochondrial respiration).
1. Immunological Methods
One method for determining the expression level of UCP-2 is to utilize antibodies that specifically bind to UCP-2 to capture UCP-2 from a sample. One such approach is the so-called "sandwich immunoassay." Such methods generally involve contacting a sample from an individual with immobilized anti-UCP-2 antibodies which capture UCP-2 from the sample to form a complex. This complex is subsequently contacted with a labeled anti-UCP-2 detection antibody that preferably recognizes a different portion of UCP-2 then the immobilized antibody. This detection antibody binds to the complex containing UCP-2 and immobilized antibody to form a ternary complex that can be quantitated based upon the magnitude of a signal generated by the labeled detection antibody. Certain of the sandwich assays are enzyme-linked immunosorbent assays (ELISA) in which the detection antibody bears an enzyme. The detection antibody is detected by providing a substrate for the enzyme to generate a detectable signal.
Further guidance regarding the methodology and steps of a variety of antibody assays is provided, for example, in U.S. Pat. No. 4,376,110 to Greene; "Immunometric Assays Using Monoclonal Antibodies," in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chap. 14 (1988); Bolton and Hunter, "Radioimmunoassay and Related Methods," in Handbook of Experimental Immunology (D. M. Weir, ed.), Vol. 1, chap. 26, Blackwell Scientific Publications, 1986; Nakamura, et al., "Enzyme Immunoassays: Heterogeneous and Homogenous Systems," in Handbook of Experimental Immunology (D. M. Weir, ed.), Vol. 1, chap. 27, Blackwell Scientific Publications, 1986; and Current Protocols in Immunology, (John E. Coligan, et al., eds), chap. 2, section I, (1991).
The antibodies used to perform the foregoing assays can include polyclonal antibodies, monoclonal antibodies and fragments thereof as described supra. Monoclonal antibodies can be prepared according to established methods (see, e.g., Kohler and Milstein (1975) Nature 256:495; and Harlow and Lane (1988) Antibodies: A Laboratory Manual (C.H.S.P., N.Y.)).
2. Activity Assays
Various different UCP-2 activities can also be determined to detect an increase in UCP-2 expression. For example, given its uncoupling role in mitochondria, certain assays involve detecting an increase in mitochondrial respiration mediated by UCP-2 in a sample from a patient potentially suffering from stroke or at risk for stroke relative to a baseline value. Assays can be conducted using isolated cells or tissue samples, or isolated mitochondrial preparations. Instead of measuring mitochondrial respiration, one can instead measure the extent of mitochondrial swelling. Methods for conducting such mitochondrial assays are known in the art and described, for example, by Salvioli et al. (1997) FEBS Lett 411:77-82; and Smiley et al. (1991) Proc. Natl. Acad. Sci. USA 88:3671-3675). Methods for conducting such assays with certain uncoupling proteins is discussed, for example, in PCT publications WO 00/17353 and WO 98/45313.
By analogy to UCP-1 activity, another activity that can serve as a measure of UCP-2 activity in some instances is the transport of fatty acids by UCP-2. UCP-1 proton transport activity is regulated by fatty acids. In vitro studies also show that UCP-1 can function as a fatty acid anion transporter. It is believed that fatty acids stimulate proton transport across the mitochondrial membrane by themselves mediating the transport of protons as UCP-1 protonophores (see, e.g., Garlid, K. D., et al. (1996) J. Biol. Chem. 271:2615-2620). The sequence homology between UCP-1 and UCP-2 indicates that UCP-2 activity also includes fatty acid transport. Such assays can be conducted using labeled (e.g., radiolabeled) fatty acids.
C. Time Course Analyses
Certain prognostic methods of assessing a patient's risk of stroke involve monitoring UCP-2 expression levels for a patient susceptible to stroke to track whether there appears to be an increase in UCP-2 expression over time. An increase in UCP-2 expression over time can indicate that the individual is at increased risk for stroke. As with other measures of UCP-2, the UCP-2 expression level for the patient at risk for stroke is compared against a baseline value (see infra). The baseline in such analyses can be a prior value determined for the same individual or a statistical value (e.g., mean or average) determined for a control group (e.g., a population of individuals with no apparent neurological risk factors). An individual showing a statistically significant increase in UCP-2 expression levels over time can prompt the individual's physician to take prophylactic measures to lessen the individual's potential for stroke. For example, the physician can recommend certain life style changes (e.g., improved diet, exercise program) to reduce the risk of stroke. Alternatively, or in addition, the physician can prescribe medicines to reduce the stroke risk.
The various test values determined for a sample from an individual believed to have suffered a stroke or to be susceptible to stroke typically are compared against a baseline value to assess the extent of increased UCP-2 expression, if any. This baseline value can be any of a number of different values. In some instances, the baseline value is a value established in a trial using a healthy cell or tissue sample that is run in parallel with the test sample. Alternatively, the baseline value can be a statistical value (e.g., a mean or average) established from a population of control cells or individuals. For example, the baseline value can be a value or range which is characteristic of a control individual or control population. For instance, the baseline value can be a statistical value or range that is reflective of UCP-2 levels for the general population, or more specifically, healthy individuals not susceptible to stroke. Individuals not susceptible to stroke generally refer to those having no apparent risk factors correlated with stroke, such as high blood pressure, high cholesterol levels, diabetes, smoking and high salt diet, for example.
Samples can be obtained from a variety of sources. For example, since the methods are designed primarily to diagnosis and assess risk factors for humans to neurological disorders such as stroke, samples are typically obtained from a human subject. However, the methods can also be utilized with samples obtained from all other mammals, such as non-human primates (e.g., apes and chimpanzees), mice and rats. Such samples can be referred to as a patient sample or a biological sample.
Samples can be obtained from the tissues or fluids of an individual, as well as from cell cultures or tissue homogenates. For example, samples can be obtained from whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, spinal fluid and amniotic fluid. Samples can also be derived from in vitro cell cultures, including the growth medium, recombinant cells and cell components.
Because certain diagnostic methods involve evaluating the level of expression in nerve cells, the sample can be obtained from various types of nerve cells including, but not limited to, neuron cells, glial cells, microglial cells and cortical neuron cells. Current evidence indicates that one consequence of stroke is that the blood/brain barrier becomes more permeable. Stroke also results in the death of certain cells which, upon dying, are lysed, thus expelling cellular components such as UCP-2. These components can then traverse the blood/brain barrier and be picked up by the circulatory system. Consequently, certain diagnostic and prognostic methods are conducted with blood samples.
Because UCP-2 is expressed as part of a neuroprotective response, diagnostic samples are collected any time after an individual is suspected to have had a stroke or to exhibit symptoms that are predictors of stroke. In prophylactic testing, samples can be obtained from an individual who present with risk factors that indicate a susceptibility to stroke (e.g., high blood pressure, obesity, diabetes) as part of a routine assessment of the individual's health status.
Some of the diagnostic and prognostic methods that involve the detection of UCP-2 transcript begin with the lysis of cells and subsequent purification of nucleic acids from other cellular material. To measure the transcription level (and thereby the expression level) of UCP-2, a nucleic acid sample comprising mRNA transcript(s) of UCP-2, or nucleic acids derived from the mRNA transcript(s) is obtained. A nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript, or a subsequence thereof, has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, suitable samples include, but are not limited to, mRNA transcripts of UCP-2, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from UCP-2 nucleic acids, and RNA transcribed from amplified DNA.
In some methods, a nucleic acid sample is the total mRNA isolated from a biological sample; in other instances, the nucleic acid sample is the total RNA from a sample taken from an individual. Any RNA isolation technique such as those described supra that do not select against the isolation of mRNA can be utilized for the purification of such RNA samples. If needed to improve the detection limits of the method, UCP-2 can be amplified prior to further analysis using established amplification techniques such as those described above.
IV. Therapeutic/Prophylactic Treatment Methods
The upregulation of UCP-2 detected in the neuroprotection model system indicates that methods that increase the expression or activity of UCP-2 can be utilized in treating individuals that have suffered a neuronal injury, as well as prophylactically treating individuals at risk for neuronal injury. In general, such methods involve administering to an individual that has suffered a neurological injury or that is at risk for such injury, an agent in an amount effective to increase the expression or activity of UCP-2. The neurological injury being treated can include, stroke (particularly ischemic stroke), and all other neurological diseases associated with altered mitochondrial function including, but not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, inherited ataxias, schizophrenia, dementia, mitochondrial encephalopathy, amylotrophic lateral sclerosis, motor neuron diseases and others (see, e.g., Beal (2000) TINS 23: 298). In a broader view, mitochondrial dysfunction is a critical factor in cell death by necrosis and apoptosis. Thus, many diseases (neurological and peripheral) involving cell death by apoptosis and/or necrosis can be targeted by an increase in UCP-2 activity (e.g., myocardial ischemia, diabetes, hepatic cierrosis, muscular dystrophies, spinal cord injuries).
Therapeutic/prophylactic intervention to increase UCP-2 expression and/or activity include but are not limited to administration of UCP-2 inducers shortly after an ischemic episode, and chronic administration in individuals with a previous stroke, at higher risk for stroke, and in genetically predisposed individuals.
Depending upon the individual's condition, the agent can be administered in a therapeutic or prophylactic amount. If the individual has suffered a neurological injury, then, at least for some period of time after the injury, the agent is typically administered in a therapeutic amount. A "therapeutic amount," as defined herein, means an amount sufficient to remedy a neurological disease state or symptoms, or otherwise prevent, hinder, retard or reverse the progression of a neurological disease or any other undesirable symptoms, especially stroke and more particularly ischemic stroke. If, however, the individual only presents with risk factors suggesting he or she is susceptible to neurological injury, then the agent is administered in a prophylactically effective amount. A prophylactic amount can also be administered as part of a long-term regimen for individuals that have already had a stroke and are at increased risk of another stroke. A "prophylactic amount" is an amount sufficient to prevent, hinder or retard a neurological disease or any undesirable symptom, particularly with regard to neurological disorders such as stroke, particularly ischemic stroke.
Prophylactic treatment can commence whenever an individual is at increased risk of suffering from a neurological disorder such as stroke. For example, individuals having risk factors known to be correlated with stroke can be administered prophylactic amounts of an agent that increases UCP-2 activity. Examples of such individuals include those that: are overweight or obese, have high blood pressure, have elevated cholesterol levels, have diabetes and/or are about to undergo medial treatment that puts the individual at risk (e.g., a patient about to undergo cardiac by-pass surgery).
In view of UCP-2 activity as a mitochondrial protein that regulates mitochondrial permeability transition, agents utilized in therapeutic methods can include those that affect the mitochondrial transition pore in a similar manner. Similarly, given the evidence indicating that UCP-2 inhibits apoptosis at least in part by inhibiting caspase 3 activation, therapeutic agents can also include agents with similar inhibitory properties.
B. UCP-2 and Other Agents
A variety of different agents can be administered to achieve the desired increase in UCP-2 activity. In some instances, the agent is a purified UCP-2 polypeptide as defined supra, including active fragments thereof. Methods for preparing purified UCP-2 are described infra. Other therapeutic agents that are administered act to stimulate the synthesis or expression of UCP-2. Such agents include those that induce the UCP-2 promoter, for example, thereby increasing expression of UCP-2 in cells. Compounds having such activity can be identified using the screening methods described below in the screening section. Often such compounds are administered in combination with a pharmaceutically-acceptable carrier. Such carriers and modes of administration are discussed further in the section on pharmaceutical compositions infra. Various inducers of UCP-2 can be utilized in certain methods. Specific examples of such inducers include PPAR.gamma. agonists such as .beta.3-agonists such as isoproterenol. Inducers can also include agents that activate the transcription of UCP-2.
Compounds increasing UCP-2 activity can be administered in combination with various other compounds. For example, the compound can be administered with an agent that increases the permeability of the blood/brain barrier to facilitate delivery of the agent activating UCP-2 activity to the brain. Such agents include, but are not limited to, bradykinin, serotonin, histamine and arachidonic acid. Other compounds that can be administered with the compound increasing UCP-2 activity include compounds that protect against clotting and prevent thrombus formation including but not limited to heparin and fucoidan.
Because UCP-2 is transmembrane protein of the inner mitochondrial membrane that functions as a proton channel (Ricquier and Bouillaud (2000) Biochem J 345: 161-179), identification of agonists that increase UCP-2 activity are another therapeutic option (see, generally, Drews (2000) Science 287: 1960 for a discussion of drug targets to ion channels). These agonists can gate the UCP-2 channel in the absence of physiological regulation and gating mechanisms and lead to an increase in UCP-2-mediated proton flow.
Additional agents that can be administered in combination with the identified compounds and delivery mode options are discussed in detail in the pharmaceutical composition section infra.
C. Gene Therapy
Gene therapy is another option for increasing UCP-2 expression. Such methods generally involve administering to an individual a nucleic acid molecule that encodes UCP-2 or an active fragment thereof. The administered nucleic acid increases the level of UCP-2 expression in one or more tissues, especially nerve cells, and particularly neuron cells. The nucleic acid is administered to achieve synthesis of UCP-2 in an amount effective to obtain a therapeutic or prophylactic effect in the individual receiving the therapy. As used herein, the term "gene therapy" refers to therapies in which a lasting effect is obtained with a single treatment, and methods wherein the gene therapeutic agents are administered multiple times to achieve or maintain the desired increase in UCP-2 expression.
The nucleic acid molecules encoding UCP-2 can be administered ex vivo or in vivo. Ex vivo gene therapy methods involve administering the nucleic acid to cells in vitro and then transplanting the cells containing the introduced nucleic acid back into the individual being treated. Techniques suitable for the in vitro transfer of UCP-2 nucleic acids into mammalian cells include, but are not limited to, the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran and calcium phosphate precipitation methods. Once the cells have been transfected, they are subsequently introduced into the patient.
Certain ex vivo methods are based on the use of any form of genetically-modified neuronal stem cells for the continuous intracerebral delivery of UCP-2. For example, UCP-2 producing cells can be implanted or surrounded by a semipermeable membrane (e.g., a capsule), directly into the intracerebroventricular space or into the cerebrospinal fluid.
In vivo gene therapy methods involve the direct administration of nucleic acid or a nucleic acid/protein complex into the individual being treated. In vivo administration can be accomplished according to a number of established techniques including, but not limited to, injection of naked nucleic acid, viral infection, transport via liposomes and transport by endocytosis. Of these, transfection with viral vectors and viral coat protein-liposome mediated transfection are commonly used methods (see, e.g., Dzau et al (1993) Trends in Biotechnology 11:205-210). Suitable viral vectors include, for example, adenovirus, adeno-associated virus and retrovirus vectors.
Methods can be designed to selectively deliver nucleic acids to certain cells. Examples of such cells include, neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells. Because UCP-2 exhibits a neuroprotective effect, certain treatment methods are designed to selectively express UCP-2 in neuron cells and/or target the nucleic acid for delivery to nerve cells. However, in other instances non-nerve cells are targeted (see, e.g., microglia, astrocytes, endothelial cells, oligodendrocytes). One technique for achieving selective expression in nerve cells is to operably link the nucleic acid encoding UCP-2 to a promoter that is primarily active in nerve cells. Examples of such promoters include, but are not limited to, prion protein promoter, calcium-calmodulin dependent protein kinase promoter, enolase promoter and PDGF.beta.-promoter. Alternatively, or in addition, the nucleic acid can be administered with an agent that targets the nucleic acid to nerve cells. For instance, the nucleic acid can be administered with an antibody that specifically binds to a cell-surface antigen on the nerve cells or a ligand for a receptor on neuronal cells. When liposomes are utilized, substrates that bind to a cell-surface membrane protein associated with endocytosis can be attached to the liposome to target the liposome to nerve cells and to facilitate uptake. Examples of proteins that can be attached include capsid proteins or fragments thereof that bind to nerve cells, antibodies that specifically bind to cell-surface proteins on nerve cells that undergo internalization in cycling and proteins that target intracellular localizations within nerve cells (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432; and Wagner, et al. (1990) Proc. Natl. Acad. Sci. USA 87:3410-3414). Gene marking and gene therapy protocols are reviewed by Anderson et al. (1992) Science 256:808-813.
Various other delivery options can also be utilized. For instance, a nucleic acid containing UCP-2 (e.g., a vector containing UCP-2) can be injected directly into the cerebrospinal fluid. Alternatively, such nucleic acids can be administered by intraventricular injections.
V. Screening Methods
A number of different screening protocols can be utilized to identify agents that increase the level of expression of UCP-2 in cells, particularly mammalian cells, especially human cells. In general terms, the screening methods involve screening a plurality of agents to identify an agent that increases the activity of UCP-2 by binding to UCP-2, preventing an inhibitor from binding to UCP-2 or activating expression of UCP-2, for example.
A. UCP-2 Binding Assays
Preliminary screens can be conducted by screening for compounds capable of binding to UCP-2, as at least some of the compounds so identified are likely UCP-2 activators. The binding assays usually involve contacting a UCP-2 protein with one or more test compounds and allowing sufficient time for the protein and test compounds to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet, J. P. and Yamamura, H. I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp. 61-89. The UCP-2 protein utilized in such assays can be naturally expressed, cloned or synthesized UCP-2.
B. Expression Assays
Certain screening methods involve screening for a compound that up-regulates the expression of UCP-2. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing UCP-2 and then detecting and an increase in UCP-2 expression (either transcript or translation product). Some assays are performed with neuron cells that express endogenous UCP-2 (e.g., cortical neuron cells, glial cells or microglial cells). Other expression assays are conducted with non-neuronal cells that express an exogenous UCP-2.
UCP-2 expression can be detected in a number of different ways. As described infra, the expression level of UCP-2 in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of UCP-2. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in situ-hybridization techniques (see above). Alternatively, UCP-2 protein can be detected using immunological methods in which a cell lysate is probe with antibodies that specifically bind to UCP-2.
Other cell-based assays are reporter assays conducted with cells that do not express UCP-2. Certain of these assays are conducted with a heterologous nucleic acid construct that includes a UCP-2 promoter that is operably linked to a reporter gene that encodes a detectable product. Suitable UCP-2 promoters are described, for example, in PCT Publication WO 00039315. A number of different reporter genes can be utilized. Some reporters are inherently detectable. An example of such a reporter is green fluorescent protein that emits fluorescence that can be detected with a fluorescence detector. Other reporters generate a detectable product. Often such reporters are enzymes. Exemplary enzyme reporters include, but are not limited to, .beta.-glucuronidase, CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature 282:864-869), luciferase, .beta.-galactosidase and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
In these assays, cells harboring the reporter construct are contacted with a test compound. A test compound that either activates the promoter by binding to it or triggers a cascade that produces a molecule that activates the promoter causes expression of the detectable reporter. Certain other reporter assays are conducted with cells that harbor a heterologous construct that includes a transcriptional control element that activates expression of UCP-2 and a reporter operably linked thereto. Here, too, an agent that binds to the transcriptional control element to activate expression of the reporter or that triggers the formation of an agent that binds to the transcriptional control element to activate reporter expression, can be identified by the generation of signal associated with reporter expression.
The level of expression or activity can be compared to a baseline value. As indicated above, the baseline value can be a value for a control sample or a statistical value that is representative of UCP-2 expression levels for a control population (e.g., healthy individuals not at risk for neurological injury such as stroke). Expression levels can also be determined for cells that do not express UCP-2 as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
A variety of different types of cells can be utilized in the reporter assays. As stated above, certain cells are nerve cells that express an endogenous UCP-2. Cells not expressing UCP-2 can be prokaryotic, but preferably are eukaryotic. The eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs. Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cell lines.
Various controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.
C. Assays of UCP-2 Activity
Various screening methods can be conducted to identify compounds that increase the activity of UCP-2. Some of the UCP-2 activities that can be measured include determination of mitochondrial respiration and fatty acid transport rates as described supra in the section on diagnostic methods. The sequence homology between UCP-2 and UCP-1 indicates that under some conditions UCP-2 proton transport can be inhibited by certain purine nucleotides, such as diphosphate and triphosphate purine nucleotides. GDP, for instance, has be shown to be an inhibitor that binds to an inhibitory site on UCP-1 (see, e.g., Murdza-Inglis, D. L., et al. (1994) J. Biol. Chem. 269:7435-38; and Bouillaud, F., et al. (1994) EMBO J. 13:1990-97). Thus, screens to identify compounds that inhibit binding of such purine nucleotides either by binding to the same inhibitory site or at another site of UCP-2 can serve as potential activators of UCP-2. These type of compounds can be identified by using labeled-purine nucleotides, for example, and detecting the ability of test compounds to inhibit binding of the labeled nucleotides to UCP-2 (e.g., UCP-2 containing mitochondrial membrane preparations). Assays based on measuring the mitochondrial membrane potential, and the associated protonmotive force (PMF), can be performed in both yeast and mammalian cells upon ectopic expression of UCP-2. Compounds that influence the PMF can be subsequently identified by fluorescent dyes or electrochemical methods.
Other assays can also be utilized in the screening process. Examples include assaying mitochondrial respiration rates, mitochondrial swelling and/or transport of fatty acids as described supra in the section diagnostic and prognostic methods. Regardless of the particular assay, various controls can be conducted to ensure that the observed activity is genuine. For example, assays can be conducted with cells that do not express UCP-2 or assays can be conducted in which cells that do express UCP-2 are not contacted with test compound.
Compounds that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models such as the rat model system described infra in Example 1. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if UCP-2 is in fact upregulated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice and rats.
Certain methods are designed to test not only the ability of a lead compound to increase UCP-2 activity in an animal model, but to provide protection after the animal has undergone transient ischemia for a longer period of time than shown to provide a protective effect. In such methods, a lead compound is administered to the model animal (i.e., an animal, typically a mammal, other than a human). The animal is subsequently subjected to transient ischemia for a period longer in duration than that shown to provide a protective effect. The conditions causing the ischemia are halted and UCP-2 activity monitored to identify those compounds still able to increase UCP-2 activity above a baseline level. Compounds able to enhance UCP-2 expression beyond the time period in which UCP-2 is upregulated in preconditioning models are good candidates for further study.
E. Compounds Affecting Mitochondria and Cell Apoptosis
Because of the evidence indicating that UCP-2 affects cellular apoptosis by altering mitochondrial permeability transition and membrane potential, as well as inhibiting activation of caspase-3 activation, screens can also be conducted to identify compounds that have similar effects on mitochondrial permeability transition, membrane potential and caspase-3 activation.
A variety of methods can be utilized to determine mitochondrial membrane potentials. One approach is to utilize fluorescent indicators(see, e.g., Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals, 6th ed., Molecular Probes, OR, pp. 266-274 and 589-594). Various non-fluorescent probes can also be utilized (see, e.g., Kamo et al. (1979) J. Membrane Biol. 49:105). Mitochondrial membrane potentials can also be determined indirectly from mitochondrial membrane permeability (see, e.g., Quinn (1976) The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Md., pp. 200-217). Various ion sensitive electrode can also be utilized.
Caspase 3 activity can be monitored utilizing various known substrates known in the art. Suitable caspase-3 assays are described, for example, by (Ellerby et al., (1997) J. Neurosci. 17:6165; Rosen et al., (1997) J. Cell. Biochem. 64:50; and Kluck et al. (1997) Science 275:1132). Another caspase assay is described in Example 4 below.
Cytochrome c release from mitochondria can be detected using any of a number of immunological or spectroscopic methods.
F. Test Compounds
The screening methods can be conducted with essentially any type of compound potentially capable of activating UCP-2 expression. Consequently, test compounds can be of a variety of general types including, but not limited to, polypeptides; carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; or amino acid analogs. The test compounds can be of a variety of chemical types including, but not limited to, heterocyclic compounds, carbocyclic compounds, .beta.-lactams, polycarbamates, oligomeric-N-substituted glycines, benzodiazepines, thiazolidinones and imidizolidinones. Certain test agents are small molecules, including synthesized organic compounds.
Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries, for example. A number of different types of combinatorial libraries and methods for preparing such libraries have been described, including for example, PCT publications WO 93/06121, WO 95/12608, WO 95/35503, WO 94/08051 and WO 95/30642, each of which is incorporated herein by reference.
VI. Production of UCP-2
A. UCP-2 Nucleic Acids
UCP-2 nucleic acids can be obtained by any suitable method known in the art, including, for example: (1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, (2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features, (3) various amplification procedures [e.g., polymerase chain reaction (PCR)] using primers that specifically hybridize to UCP-2 nucleic acids; and 4) direct chemical synthesis.
More specifically, UCP-2 nucleic acids can be obtained using established cloning methods. The nucleotide sequence of a gene or cDNA encoding UCP-2 (see, e.g., SEQ ID NO:1) is used to provide probes that specifically hybridize to a UCP-2 cDNA in a cDNA library, a UCP-2 gene in a genomic DNA sample, or to a UCP-2 mRNA in a total RNA sample (e.g., in a Southern or Northern blot). The libraries are preferably prepared from nerve cells. Once the target nucleic acid is identified, it can be isolated and cloned using well-known amplification techniques. Such techniques include, the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q.beta.-replicase amplification, the self-sustained sequence replication system (SSR) and the transcription based amplification system (TAS). Cloning methods that can be utilized to clone UCP-2 are described in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, 152, Academic Press, Inc. San Diego, Calif.; Sambrook, et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed) Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; and Current Protocols (1994), a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc.
UCP-2 nucleic acids can also be obtained utilizing various amplification techniques. Such methods include, those described, for example, in U.S. Pat. No. 4,683,202 to Mullis et al.; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990); Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117.
As an alternative to cloning a nucleic acid, a suitable nucleic acid can be chemically synthesized. Direct chemical synthesis methods include, for example, the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. While chemical synthesis of DNA is often limited to sequences of about 100 bases, longer sequences can be obtained by the ligation of shorter sequences. Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce a UCP-2 sequence.
Further specific guidance regarding the preparation of UCP-2 nucleic acids is provided by Fleury et al. (1997) Nature Genetics 15:269-272; Tartaglia et al., PCT Publication No. WO 96/05861; and Chen, et al., PCT Publication No. WO 00/06087, each of which is incorporated herein in its entirety.
B. UCP-2 Proteins
UCP-2 proteins can be produced through isolation from natural sources, recombinant methods and chemical synthesis. For example, UCP-2 proteins can be prepared by expressing cloned UCP-2 in a host cell. Cloned UCP-2 sequences are expressed in hosts after the sequences have been operably linked to an expression control sequence in an expression vector. Expression vectors are usually replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
Typically, the polynucleotide that encodes UCP-2 is placed under the control of a promoter that is functional in the desired host cell to produce relatively large quantities of UCP-2. An extremely wide variety of promoters are well-known, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. Constructs that include one or more of these control sequences are termed "expression cassettes." Expression can be achieved in prokaryotic and eukaryotic cells utilizing promoters and other regulatory agents appropriate for the particular host cell. Exemplary host cells include, but are not limited to, E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques. A description of the preparation of the recombinant nucleic acids including sequences that encode UCP-2 can be found, for example, in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, Calif. (Berger); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement) (Ausubel).
Once expressed, the recombinant polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, ion exchange and/or size exclusivity chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)).
As an option to recombinant methods, UCP-2 can be chemically synthesized. Such methods typically include solid-state approaches, but can also utilize solution based chemistries and combinations or combinations of solid-state and solution approaches. Examples of solid-state methodologies for synthesizing proteins are described by Merrifield (1964) J. Am. Chem. Soc. 85:2149; and Houghton (1985) Proc. Natl. Acad. Sci., 82:5132. Fragments of UCP-2 can be synthesized and then joined together. Methods for conducting such reactions are described by Grant (1992) Synthetic Peptides: A User Guide, W.H. Freeman and Co., N.Y.; and in "Principles of Peptide Synthesis," (Bodansky and Trost, ed.), Springer-Verlag, Inc. N.Y., (1993).
Additional guidance specific for preparing UCP-2 proteins is provided by Fleury et al. (1997) Nature Genetics 15:269-272; Tartaglia et al., PCT Publication No. WO 96/05861; and Chen, et al., PCT Publication No. WO 00/06087.
A. Synthesis of Analogs
Active test agents identified by the screening methods described herein that increase UCP-2 activity can serve as lead compounds for the synthesis of analog compounds. Typically, the analog compounds are synthesized to have an electronic configuration and a molecular conformation similar to that of the lead compound. Identification of analog compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available. See, e.g., Rein et al., (1989) Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York).
Once analogs have been prepared, they can be screened using the methods disclosed herein to identify those analogs that exhibit an increased ability to increase UCP-2 activity. Such compounds can then be subjected to further analysis to identify those compounds that appear to have the greatest potential as pharmaceutical agents. Alternatively, analogs shown to have activity through the screening methods can serve as lead compounds in the preparation of still further analogs, which can be screened by the methods described herein. The cycle of screening, synthesizing analogs and rescreening can be repeated multiple times.
B. Pharmaceutical Compositions
Compounds identified by the screening methods described above, analogs thereof and UCP-2 itself can serve as the active ingredient in pharmaceutical compositions formulated for the treatment of various neurological disorders including stroke. The compositions can also include various other agents to enhance delivery and efficacy. For instance, compositions can include agents capable of increasing the permeability of the blood/brain barrier. Other agents that can be coadministered include anticoagulants and blood thinners. The compositions can also include various agents to enhance delivery and stability of the active ingredients.
Thus, for example, the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
The composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide (e.g., UCP-2), the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).
The pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments. Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, 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 that exhibit large therapeutic indices are preferred.
The data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.
For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.
Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged active ingredient with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the packaged active ingredient with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
Claim 1 of 8 Claims
What is claimed is:
1. A method for diagnosing occurrence of neuronal exposure to hypoxia-ischemia comprising detecting in a patient's blood, serum, or cerebrospinal fluid sample an elevated level of UCP-2 polypeptide, wherein said elevated level of UCP-2 polypeptide is diagnostic of neuronal exposure to hypoxia-ischemia.