JP2004510965A - Perilipin as a target for regulating weight, muscle mass and diabetes - Google Patents

Abstract

The present invention relates to screening assays for identifying compounds that modulate the activity or expression of perilipin. The invention also relates to methods and therapeutic compositions for preventing or treating weight loss or diabetes, comprising administering to a subject a compound that modulates the activity or expression of perilipin. In one aspect, the invention relates to methods and compositions for antagonizing the activity of perilipin for the treatment and / or prevention of obesity or diabetes, and for increasing lipid metabolism and muscle mass. In another aspect, the invention relates to methods and compositions for agonizing the activity of perilipin, with the aim of increasing lipid accumulation to gain weight.

Description

[0001]
1. Foreword
The present invention relates to perilipin as a novel target for regulating lipid homeostasis, muscle mass and body weight. The present invention relates to screening assays for identifying compounds that modulate the activity or expression of perilipin, a novel target of the present invention. The present invention also relates to methods and compositions for treating weight disorders comprising administering to a subject a compound that modulates the activity or expression of perilipin. In one embodiment, the present invention relates to methods and compositions for antagonizing perilipin activity or expression for the purpose of treating and / or preventing obesity or diabetes, and increasing lipid metabolism and muscle mass. . In another aspect, the present invention relates to methods and compositions for agonizing the activity of perilipin for increasing lipid accumulation in a subject to gain weight.
[0002]
2. Background of the Invention
In higher organisms, the primary function of adipocytes is to store excess energy as triacylglycerol in intracellular lipid droplets and release that energy as fatty acids when needed (Yeaman et al., 1990, Biochim. Biophys. Acta 1052: 128-132). Despite the wealth of information on the metabolism of carbohydrate stores, especially glycogen, the details of the mechanism by which lipid stores are packaged and hydrolyzed are not well understood. The rate-limiting step in lipolysis, the process by which triacylglycerol is hydrolyzed to fatty acids and glycerol, is catalyzed by hormone-sensitive lipase (HSL). Hormonal stimulation of HSL activity involves the transfer of this enzyme to the surface of lipid storage droplets. Perilipin is an abundant adipocyte protein and exists as three isoforms (A, B and C), transcripts that are spliced differently from a single gene. It has been speculated that under ground state, perilipin coats lipid droplets and somehow prevents HSL from gaining access to lipid droplets (Londos et al., 1995, Biochem. Soc. Trans. 23: 611-615; Londos et al., 1999, Semin. Cell. Dev. Biol. 10: 51-58; Souza et al., 1998, J. Biol. Chem. 273: 24665-24669). Lipolysis activation of adipocytes is associated with protein kinase A-mediated phosphorylation of perilipin, which alters the lipid droplet surface to reveal lipid microdroplets and reduces HSL access to core lipids. Enable (Londos et al., 1999, Semin. Cell. Dev. Biol. 10: 51-58). However, the role of perilipin in lipid homeostasis and energy metabolism in vivo is rather uncertain.
[0003]
3. Summary of the Invention
The present invention relates to perilipin as a target for regulating lipid homeostasis and for regulating muscle mass and weight gain. The present invention identifies compounds that modulate the activity and / or expression of perilipin protein as a means of identifying compounds useful for treating body weight-related disorders and / or inappropriate regulation of lipid metabolism. To perform screening assays. The invention also encompasses pharmaceutical compositions containing a compound that modulates the activity and / or expression of perilipin protein for treating a weight-related disorder and / or inappropriate regulation of lipid metabolism.
[0004]
The present invention is based, in part, on the inventors discovering a key role for perilipin protein in lipid homeostasis, muscle mass and energy metabolism in vivo. In the following examples, when the mouse perilipin gene is disrupted (plin^{− / −}Mouse), which shows that mice displaying constitutively activated hormone-sensitive lipase can be obtained. plin^{− / −}Mice can consume more food, but maintain normal weight compared to control mice. plin^{− / −}Mice exhibit increased basal lipolysis, increased metabolism and are resistant to diet-induced obesity. Furthermore, plin^{− / −}Mice gain muscle mass without exercise. These results demonstrate that perilipin is a target for regulating lipolysis and energy balance, and for anti-obesity drug administration.
[0005]
The present invention provides screening assays for identifying compounds that modulate the activity, expression, and / or phosphorylation state of a perilipin protein. In particular, the present invention relates to perilipin binding using recombinantly expressed perilipin, cells that endogenously express one or more perilipin isoforms (eg, adipocytes), or cell lines transfected with perilipin. An in vitro assay for identifying sex compounds is provided. The perilipin-transfected cell line may further include a reporter gene whose expression level is regulated by perilipin.
[0006]
The present invention provides a method of screening for an agent that interacts with a perilipin isoform or a fragment thereof, comprising: (a) contacting a perilipin isoform or a fragment thereof with a candidate agent; Determining whether the candidate agent interacts with the perilipin isoform or fragment thereof. According to these methods, perilipin isoforms or fragments thereof can be expressed endogenously from cells such as steroidogenic cells or adipocytes, and genetically engineered cells to express perilipin isoforms or fragments thereof. You can also.
[0007]
The invention also provides a method of screening for an agent that modulates expression of a perilipin isoform, the method comprising: (a) contacting a first population of cells expressing a perilipin isoform with a candidate agent. (B) contacting a second population of cells expressing the perilipin isoform with a control agent; and (c) the level of the perilipin isoform or the perilipin isoform in first and second cell populations. Comparing the level of mRNA encoding If the expression level of the perilipin isoform or the level of mRNA encoding the perilipin isoform is higher in the first cell population than in the second cell population, an agonist of perilipin has been identified. A lower level of expression of the perilipin isoform or the level of mRNA encoding the perilipin isoform in the first cell population than in the second cell population has identified an antagonist of perilipin.
[0008]
The invention also provides a method of screening for an agent that modulates the activity of a perilipin isoform, the method comprising: (a) contacting a first population of cells expressing a perilipin isoform with a candidate agent. (B) contacting a second population of cells expressing the perilipin isoform with a control agent; and (c) phosphorylation levels or cellular levels of the perilipin isoform in the first and second cell populations. Comparing the induction levels of the two messengers. A higher phosphorylation level in the first cell population than the second cell population or a higher level of induction of the cellular second messenger has identified a perilipin agonist. A lower level of phosphorylation in the first population of cells or a lower level of induction of the second cellular messenger than the second population of cells has identified a perilipin antagonist.
[0009]
The invention further relates to the occurrence, development or progression of weight, body fat, muscle mass, lipid metabolism, lipid metabolism disorders (eg, lipodystrophy), the occurrence, development or progression of weight disorders (eg, obesity), lipid deposition. A method for identifying an agent to be tested for the ability to modulate the development, development or progression of a disorder characterized by atherosclerosis (eg, atherosclerosis), or diabetes development, development or progression, comprising the steps of (a) Contacting the perilipin isoform or fragment thereof and the candidate agent with the perilipin isoform or fragment / drug complex for a time sufficient to form a perilipin isoform or fragment / drug complex; Measuring the level, so that if the measured level differs from the level measured in the absence of the candidate agent, weight, Fat, muscle mass, lipid metabolism, development, progression or progression of lipid metabolism disorders (eg, lipodystrophy), development, development or progression of body weight disorders (eg, obesity), disorders characterized by lipid deposition (eg, atheroma) Agents to be tested for their ability to modulate the onset, development or progression of atherosclerosis) or the onset, development or progression of diabetes are identified.
[0010]
The invention further relates to the occurrence, development or progression of weight, body fat, muscle mass, lipid metabolism, lipid metabolism disorders (eg, lipodystrophy), the occurrence, development or progression of weight disorders (eg, obesity), lipid deposition. A method for identifying an agent to be tested for the ability to modulate the development, development or progression of a disorder characterized by atherosclerosis (eg, atherosclerosis), or diabetes development, development or progression, comprising the steps of (a) Contacting a population of cells expressing a perilipin isoform with a candidate agent for a time sufficient to form a perilipin isoform / drug complex; and (b) the level of the perilipin isoform / drug complex. Measuring, so that if the measured level is different from the level measured in the absence of the candidate agent, weight, body fat, muscle mass, The occurrence, development or progression of quality metabolism, lipid metabolism disorders (eg, lipodystrophy), the occurrence, development or progression of weight disorders (eg, obesity), and disorders characterized by lipid deposition (eg, atherosclerosis) Agents to be tested for their ability to modulate development, development or progression, or diabetes development, development or progression, have been identified.
[0011]
The invention further relates to the occurrence, development or progression of weight, body fat, muscle mass, lipid metabolism, lipid metabolism disorders (eg, lipodystrophy), the occurrence, development or progression of weight disorders (eg, obesity), lipid deposition. A method for identifying an agent to be tested for the ability to modulate the development, development or progression of a disorder characterized by atherosclerosis (eg, atherosclerosis), or diabetes development, development or progression, comprising the steps of (a) Contacting a population of cells expressing perilipin isoform with a candidate agent, and (b) phosphorylation level of perilipin isoform, induction level of cellular second messenger, or triacylglycerol, unesterified fatty acid or measuring the level of β-hydroxybutyric acid, such that the measured level is measured in the absence of the candidate agent Weight, body fat, muscle mass, lipid metabolism, the development, development or progression of lipid metabolism disorders (eg, lipodystrophy), the development, development or progression of weight disorders (eg, obesity), Compounds to be tested for their ability to modulate the development, development or progression of a disorder characterized by deposition (eg, atherosclerosis) or diabetes development, development or progression have been identified.
[0012]
The invention also provides a method of screening or identifying an agent that modulates expression of a perilipin isoform, comprising: (a) administering a candidate agent to a first animal or group of animals; Administering a control agent to a second animal or group of animals, and (c) comparing the expression level of the perilipin isoform or the mRNA encoding the perilipin isoform in the first and second animals. Comprising. If the expression level is higher in the first animal than in the second animal, a perilipin agonist has been identified. If the expression level is lower in the first animal than in the second animal, a perilipin antagonist has been identified.
[0013]
The invention also provides a method of screening or identifying an agent that modulates one or more activities of a perilipin isoform, the method comprising: (a) administering a candidate agent to a first animal or group of animals; (B) administering a control agent to a second animal or group of animals; and (c) inducing levels of cellular second messengers, phosphorylation of perilipin isoforms, or triacylglycerol in the first and second animals. Comparing the levels of non-esterified fatty acids or β-hydroxybutyric acid. Such activity can be assessed by techniques known in the art or as described herein. If the level of induction of cellular second messengers, the level of phosphorylation of perilipin isoforms, or the level of triacylglycerol, non-esterified fatty acids or β-hydroxybutyrate is higher in the first animal than in the second animal, A pin isoform agonist has now been identified. If the level of induced cellular second messengers, the level of phosphorylation of perilipin isoforms, or the level of triacylglycerol, non-esterified fatty acids or β-hydroxybutyrate is lower in the first animal than in the second animal, A pin isoform antagonist has now been identified.
[0014]
The invention also provides a method of identifying an agent that modulates the weight, body fat or muscle mass of an animal, the method comprising: (a) binding an animal or group of animals to one or more perilipin isoforms; Administering a candidate agent that modulates the expression of one or more of the above perilipin isoforms or modulates one or more activities of one or more of the perilipin isoforms, wherein (b) the candidate agent is an untreated control animal or animal Determining whether to modulate body weight, body fat or muscle mass in the animal or group of animals as compared to the group, so that the candidate agent modulates body weight, body fat or muscle mass. Thus, agents that modulate the weight, body fat, or muscle mass of an animal have been identified. Body weight, body fat or muscle mass can be assessed using techniques known to those skilled in the art or as described herein.
[0015]
The invention also provides a method of identifying an agent that modulates the development, development or progression of diabetes in an animal, the method comprising: (a) administering to an animal or group of animals predisposed to or predisposed to diabetes; Administering a candidate agent that binds to one or more perilipin isoforms, modulates the expression of one or more perilipin isoforms, or modulates one or more activities of one or more perilipin isoforms, (b) Determining whether the candidate agent modulates blood glucose, insulin sensitivity, or one or more signs or symptoms of diabetes in the untreated control animal or group of animals as compared to the untreated control animal or group of animals. And consequently, if the candidate agent modulates blood glucose, insulin sensitivity, or one or more signs or symptoms of diabetes, Occurrence of diabetes, so that the agent that modulates the development or progression have been identified.
[0016]
The invention also provides a method of identifying an agent that modulates lipid metabolism in an animal, the method comprising: (a) providing the animal or group of animals with one or more perilipin isoforms that binds to one or more perilipin isoforms; Administering a candidate agent that modulates expression of the form or modulates one or more activities of one or more of the perilipin isoforms, wherein (b) the candidate agent is compared to an untreated control animal or group of animals. Determining whether the animal or group of animals modulates lipid metabolism, such that if the candidate agent modulates lipid metabolism, an agent that modulates lipid metabolism in the animal has been identified. Become. Lipid metabolism can be assessed by techniques known in the art or as described herein.
[0017]
The present invention further provides pharmaceutical compositions that modulate the activity, expression and / or phosphorylation state of perilipin. In particular, the pharmaceutical composition may be an agonist or antagonist of perilipin. Antagonists inhibit perilipin gene transcription by competitively inhibiting another perilipin agonist or antagonist, thereby blocking the interaction of activated perilipin with its downstream signaling pathways, and thereby inhibiting perilipin gene transcription. It can act by inhibiting the processing or translation of mRNA or by inhibiting the post-translational processing of perilipin. Agonists can act by activating and / or enhancing the natural biological effects of the perilipin signaling pathway or its expression.
[0018]
In another aspect, the invention provides methods and compositions for preventing and / or treating diseases and disorders characterized by abnormal perilipin expression and / or activity in an animal. The present invention provides a method for preventing and / or treating weight disorders in animals, preferably companion animals, livestock and poultry, more preferably humans, comprising a pharmaceutical formulation that modulates perilipin expression and / or activity. Administering to the subject. In particular, a pharmaceutical composition that increases body weight and efficiency or that reduces body weight and reduces the signs or symptoms associated with obesity is administered to a human. In addition, pharmaceutical compositions that increase body weight and efficiency, or reduce body weight and reduce obesity, can be administered to livestock and poultry.
[0019]
The present invention also provides a method for preventing and / or treating diabetes in an animal, preferably a companion animal, livestock and poultry, more preferably a human, comprising a pharmaceutical formulation that modulates perilipin expression and / or activity. Administering to the subject. In particular, a pharmaceutical composition that slows or prevents the onset, development or progression of diabetes (eg, diabetes associated or unrelated to obesity) is administered to an animal, preferably a companion animal, livestock and poultry, more preferably a human. You.
[0020]
The present invention also relates to the prevention and / or prevention of disorders of lipid metabolism (ie disorders characterized by inappropriate lipid metabolism) and disorders characterized by lipid deposition in animals, preferably companion animals, livestock and poultry, more preferably humans. Alternatively, there is provided a method of treatment, comprising administering a pharmaceutical formulation that modulates the expression and / or activity of perilipin. In particular, a pharmaceutical composition which slows or prevents the occurrence, development or progression of said disorder is administered to an animal, preferably a companion animal, livestock and poultry, more preferably a human.
[0021]
In another aspect, the invention detects and diagnoses the occurrence or progression of diseases or disorders characterized by abnormal perilipin expression and / or activity, eg, lipid metabolism disorders, weight disorders (eg, obesity), and diabetes. Alternatively, methods and compositions for monitoring are provided. In yet another aspect, the invention provides a kit comprising one or more agents identified in the screening assays of the invention and instructions for use.
[0022]
3.1. Definition
The term "Lep^{ob / ob}"" And "ob / ob" are used interchangeably herein to refer to leptin-deficient mice.
[0023]
The term "Lepr^{db / db}"" And "db / db" are used interchangeably herein to refer to leptin-resistant mice.
[0024]
The term "abnormal" as used herein with respect to perilipin expression, refers to a cell, tissue or subject in which the expression level of one or more perilipin isoforms is normal, or results in lipid metabolism disorders, weight disorders or diabetes. Means increased or decreased as compared to the expression level in cells or tissues obtained from an absent subject (ie, a reference level). The expression level of one or more perilipin isoforms can be measured by the methods described herein or known to one of skill in the art.
[0025]
As used herein, the term "abnormal" with respect to the activity of perilipin refers to a subject having a normal level of activity of one or more perilipin isoforms in cells or tissues obtained from the subject, or a disorder of lipid metabolism, weight loss or diabetes. Means an increase or decrease in the level of activity in cells or tissues obtained from a subject who is not (ie, a reference level). The activity level of one or more perilipin isoforms can be measured by the methods described herein or by methods known to one of skill in the art.
[0026]
The term "analog" as used herein with respect to a polypeptide refers to a protein that has a similar or identical function as a second polypeptide (eg, a perilipin isoform or an anti-perilipin antibody), but does not necessarily associate with the second protein. A first polypeptide that does not contain a similar or identical amino acid sequence, or does not necessarily have a similar or identical structure to a second protein. A polypeptide having a similar amino acid sequence refers to a polypeptide that meets at least one of the following: That is, (a) at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% with a second polypeptide (eg, a perilipin isoform or anti-perilipin antibody). A first polypeptide having an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical; (b) at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 consecutive amino acid residues, small At least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues A second polypeptide (eg, a perilipin isoform or anti-perilipin antibody) of at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues. A first polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to the encoding nucleotide sequence; and (c) a second polypeptide (eg, a perilipin isoform or an anti-perilipin antibody) Nucleotide sequence and at least 30 At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least The first polypeptide encoded by a nucleotide sequence that is 95%, or at least 99%, identical. A first polypeptide having a similar structure to the second polypeptide refers to a first polypeptide having a secondary, tertiary or quaternary structure similar to the second polypeptide. The structure of the polypeptide can be determined by methods known to those of skill in the art, including but not limited to peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, crystallography electron microscopy. It is possible.
[0027]
To determine the percent identity of two amino acid or nucleic acid sequences, the sequences are aligned for optimal alignment (e.g., first sequence for optimal alignment with a second amino acid or nucleic acid sequence). To introduce gaps in the amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the same position shared by the sequences (% identity = number of identical positions / total number of positions (eg, overlapping positions) × 100). In one embodiment, the two sequences are of the same length.
[0028]
The percent identity between two sequences can be determined using a mathematical algorithm. Preferred examples of mathematical algorithms used to compare two sequences include, but are not limited to, Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268, by the method of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is described in Altschul et al., (1990) J. Am. Mol. Biol. 215: 403-410, incorporated into the NBLAST and XBLAST programs. To obtain a nucleotide sequence homologous to a nucleic acid molecule of the present invention, a BLAST nucleotide search is performed using the NBLAST program, score = 100, wordlength = 12. To obtain an amino acid sequence homologous to the protein molecules of the present invention, a BLAST protein search can be performed using the XBLAST program, score = 50, wordlength = 3. To obtain a gapped alignment for comparison, Gapped BLAST can be performed as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When using BLAST, Gapped BLAST, and PSI-Blast programs, it is possible to use the default parameters of the respective programs (for example, XBLAST and NBLAST). http: // www. ncbi. nlm. nih. See gov.
[0029]
Another preferred example of a mathematical algorithm utilized for sequence comparison is the algorithm described in Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Other algorithms for sequence analysis are known in the art and are described, for example, in Torellis and Robotti (1994) Comput. Appl. Biosci. , 10: 3-5, and Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-8. The ktup included in FASTA is a control option for setting the sensitivity and speed of the search. If ktup = 2, similar regions in the two sequences to be compared are found by looking at pairs of aligned residues. When ktup = 1, a single aligned amino acid is considered. In the case of a protein sequence, ktup can be designated as 2 or 1, and in the case of DNA, ktup can be designated as 1-6. The default if ktup is not specified is 2 for proteins and 6 for DNA. For a further description of the FASTA parameters, see http: // bioweb. pasteur. fr / docs / man / man / fasta. 1. See html # sect2. The contents of which are incorporated herein by reference.
[0030]
The percent identity between two sequences can be determined using techniques similar to the above, with or without gaps. To calculate percent identity, generally the exact matches are counted.
[0031]
The term “derivative” as used herein with respect to a polypeptide includes the amino acid sequence of a second polypeptide (eg, a perilipin isoform or an anti-perilipin antibody), but wherein the substitution, deletion or addition of amino acid residues is performed. Or a first polypeptide that has been modified by the covalent attachment of any type of molecule to the second polypeptide. For example, and without limitation, polypeptides can be modified by proteolytic cleavage, binding to cellular ligands or other proteins, and the like. Derivatives of a polypeptide can be modified by chemical modification (eg, acylation, phosphorylation, carboxylation, glycosylation, selenium modification, and sulphation) using techniques known to those skilled in the art. Further, a derivative of a polypeptide may include one or more non-classical amino acids. The function of a polypeptide derivative may or may not be the same or similar to the polypeptide from which it is derived. In certain embodiments, a derivative of a perilipin isoform retains at least one function of the perilipin isoform from which it is derived. In other specific embodiments, the derivative of perilipin isoform does not possess any function of the perilipin isoform from which it is derived.
[0032]
As used herein, the term "fragment" refers to at least 5 contiguous amino acid residues of the amino acid sequence of another peptide or polypeptide, preferably at least 10, at least 15, at least 20, at least 25 , At least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, A peptide or polypeptide comprising an amino acid sequence consisting of at least 275, at least 300, at least 325, at least 350, at least 375, at least 400 or more consecutive amino acid residues. A fragment of a polypeptide may or may not have the functional activity of the polypeptide. In certain embodiments, a fragment of a perilipin isoform retains at least one function of the perilipin isoform. In other specific embodiments, a fragment of a perilipin isoform does not possess any function of the perilipin isoform from which it is derived.
[0033]
As used herein, the term "functional fragment" refers to at least 5 contiguous amino acid residues of the amino acid sequence of another peptide or polypeptide, preferably at least 10, at least 15, at least 20, At least 25, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250 An amino acid sequence consisting of at least 275, at least 300, at least 325, at least 350, at least 375, at least 400 or more contiguous amino acid residues, and of said another peptide or polypeptide. function Other refers to fragments of a peptide or polypeptide that is at least one possess activity. For example, a functional fragment of a perilipin isoform can possess the ability to bind anti-perilipin antibodies, bind lipid droplets, interact with PKA, or interact with protein phosphatase 1.
[0034]
The term "fusion protein" as used herein refers to the amino acid sequence of (i) the amino acid sequence of a first polypeptide (eg, a perilipin isoform or a fragment thereof), and (ii) the amino acid sequence of a second heterologous polypeptide. And polypeptides comprising: As used herein, the term “perilipin fusion protein” refers to the amino acid sequence of (i) the amino acid sequence of a perilipin isoform or a fragment thereof, and (ii) the amino acid sequence of a heterologous polypeptide (ie, a non-perilipin polypeptide or a fragment thereof). And polypeptides comprising: In one embodiment, the perilipin fusion protein comprises a perilipin isoform or a fragment thereof and a domain such as glutathione-S-transferase. In another embodiment, the perilipin fusion protein comprises a perilipin isoform or fragment thereof and a fragment of an antibody (preferably, the Fc domain of an antibody). Perilipin fusion proteins can be prepared using techniques well known to those skilled in the art. It is also possible to produce the fusion protein by standard recombinant DNA techniques.
[0035]
As used herein, the term "hybridizes under conditions" typically refers to nucleotide sequences that are at least 60% (65%, 70%, and preferably 75%) identical to each other are hybridized to each other. It refers to the conditions of hybridization and washing that exist in a state where they have been used. Such stringent conditions are known to those of skill in the art and are described in Current Protocols in Molecular Biology, John Wiley & Sons, NJ. Y. (1989), 6.3.1-6.3.6. As one non-limiting example, stringent hybridization conditions include hybridization at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC), followed by hybridization in 0.1 × SSC, 0.2% SDS. Consisting of one or more washes at 68 ° C. A preferred non-limiting example of stringent hybridization conditions is hybridization at about 45 ° C. in 6 × SSC, followed by one or more washes in 0.2 × SSC, 0.1% SDS at 50-65 ° C. (Ie, one or more washes at 50 ° C., 55 ° C., 60 ° C., or 65 ° C.). It will be appreciated that the nucleic acids of the present invention do not include nucleic acid molecules that hybridize under these conditions to nucleotide sequences consisting solely of A or T nucleotides. In certain embodiments, it is preferred that the polypeptide hybridizes to its full length with the perilipin isoform, and that the polypeptide has at least one function or activity of the perilipin isoform.
[0036]
As used herein, the term "isoform" refers to a variant of perilipin that is encoded by the same gene but differs in amino acid composition. There are three known isoforms of perilipin, perilipin A, perilipin B, and perilipin C.
[0037]
As used herein to describe a nucleic acid molecule or nucleotide sequence, "isolated" or "purified" refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Nucleic acid molecule or nucleotide sequence. Preferably, an “isolated” nucleic acid molecule is a sequence that is adjacent to the nucleic acid in the genomic DNA of the organism from which it originated (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid) (preferably Does not include a protein-encoding sequence). Further, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular materials and media when produced recombinantly, and contains chemical precursors and chemicals when chemically synthesized. Substantially free of other chemicals.
[0038]
“Isolated” or “purified” as used herein to describe a protein or a bioactive portion thereof (ie, a polypeptide, peptide or amino acid fragment) refers to a protein or a bioactive portion thereof. Contains substantially no cellular material or other contaminating proteins derived from the cell or tissue source from which the protein is derived, or substantially contains chemical precursors and other chemicals when chemically synthesized. Means that it is not included. As a protein or a bioactive portion thereof (ie, a polypeptide, peptide or amino acid fragment) substantially free of cellular material, the content of a heterologous protein (also referred to herein as a contaminating protein) is about 30%, 20%, 10%, or 10%. % Or less than 5% (by dry weight) protein preparation.
[0039]
The term "modulate" as used herein with respect to perilipin expression or activity, refers to any change in perilipin expression or activity (eg, up-regulation or down-regulation). Based on the present disclosure, such modulation can be measured using assays known to those of skill in the art or described herein.
[0040]
As used herein, the terms “nucleic acid” and “nucleotide sequence” refer to DNA molecules (eg, cDNA, genomic DNA), RNA molecules (eg, mRNA), a combination of DNA and RNA molecules, ie, hybrid DNA / RNA. Molecules and analogs of DNA or RNA molecules. Such analogs can be made using nucleotide analogs, including but not limited to inosine or tritylated bases. In addition, such analogs also include DNA or RNA molecules that contain a modified backbone that confers beneficial attributes to the molecule, such as, for example, nuclease resistance or increased ability to cross cell membranes. The nucleic acid or nucleotide sequence may be single-stranded or double-stranded, and may contain both single- and double-stranded portions, or may contain triple-stranded portions, but is preferably double-stranded. It is a single-stranded DNA. In one embodiment, the nucleotide sequence comprises a continuous open reading frame encoding a perilipin isoform or a fragment thereof, for example, a cDNA molecule.
[0041]
As used herein, the terms “prevent” and “prevention” refer to weight disorders (eg, obesity), diabetes (type 1 or type 2), or disorders characterized by lipid deposition (eg, atherosclerosis). Disease), or preventing the occurrence or recurrence of one or more signs or symptoms associated with lipid metabolism disorders (eg, lipodystrophy).
[0042]
As used herein, a “prophylactically effective amount” is characterized by a disorder of weight (eg, obesity), diabetes (type 1 or type 2), a disorder of lipid metabolism (eg, lipodystrophy), or lipid deposition. Modulates the expression and / or activity of one or more perilipin isoforms sufficient to prevent the occurrence or recurrence of one or more signs or symptoms associated with the disorder of interest (eg, atherosclerosis) An amount of an agent, or an amount of a composition comprising an agent that modulates the expression and / or activity of one or more perilipin isoforms.
[0043]
As used herein, the terms "treat" and "treatment" refer to a weight disorder (eg, obesity), diabetes (type 1 or type 2), a lipid metabolism disorder (eg, lipodystrophy), or lipid deposition. Is characterized by a reduction in the severity of one or more signs or symptoms associated with a disorder characterized by (eg, atherosclerosis); a reduction in insulin resistance in the animal; an improvement in insulin secretion in the animal; Weight loss in animals with weight loss (eg, obesity); weight gain in animals with weight loss (eg, anorexia, cachexia) characterized by underweight; increased muscle mass in the animal; Increased lipid metabolism in animals; refers to decreased glucose intolerance in animals.
[0044]
As used herein, a "therapeutically effective amount" is characterized by a weight disorder (eg, obesity), diabetes (type 1 or type 2), a lipid metabolism disorder (eg, lipodystrophy), or lipid deposition. Reduce the severity of one or more of the signs or symptoms associated with the disorder (e.g., atherosclerosis); reduce insulin resistance in the animal; improve insulin secretion in the animal; Decrease the weight of animals that have a weight disorder (eg, obesity); increase the weight of animals that have a weight disorder (eg, anorexia, cachexia) characterized by underweight; Expression of one or more perilipin isoforms sufficient to increase muscle mass; increase lipid metabolism in the animal; reduce glucose intolerance in the animal; Or it refers to the amount of activity modulate amounts of drug or expression of one or more of peri Li pins isoforms and / or activity of a composition comprising an agent that modulates,.
[0045]
4. Description of the drawings
1A-1C. @Plin^{− / −}Production of mouse. A. Targeting strategy. Exons 1 to 6 are represented by black boxes. Exon 2 and part of intron 2 between the BstXI and ApaI sites were replaced with IRES-β-galactosidase and neo.^rA replacement vector to be replaced with a gene. Exon 2 interruption occurs at codon 183 at the BstXI site. A thymidine kinase cassette (TK) was linked to the 3 'end of this targeting vector. H, HindIII; Xb, XbaI; Ap, ApaI; BX, BstXI; TK, thymidine kinase. B.ザ ン Southern blotting of tail DNA after XbaI digestion. Using the 3 'DNA probe (see A above), an 8.5-kb band containing exons 2 to 6 was detected in wild-type DNA, and plin was detected.^{− / −}In the DNA, a 7.0-kb band containing a part of the neo gene and exons 3 to 6 is detected. C.ウ ェ ス タ ン Western blotting of cell extracts from epididymis and subcutaneous fat (left) and testis (right). Perilipins A and B are detected in adipose tissue and perilipins A and C are detected in testis in wild type (Londos et al., 1999, Semin. Cell Dev. Biol. 19: 51-58). These isoforms are all products of the plin gene,^{− / −}Not detectable in sample. In wild-type mice, perilipin expression is higher in adipose tissue than in testis.
[0046]
2A-2F. Inactivation of perilipin is plin^{− / −}Phenotypic effects on body weight, fat accumulation, lipid content, and muscle mass in mice. A. Male plin^{+ / +}(N = 11), plin^+/-(N = 15), and plin^{− / −}(N = 8) mouse weight. B. Female plin^{+ / +}(N = 6), plin^+/-(N = 24), and plin^{− / −}(N = 12) mouse weight. C. @Plin^{− / −}And plin^{+ / +}Fat accumulation in mice. D. @Plin^{+ / +}And plin^{− / −}Total carcass lipid content of mice. E. FIG. @Plin^{+ / +}And plin^{− / −}Total carcass triacylglycerol content of mice. F. @Plin^{+ / +}And plin^{− / −}Total carcass protein content of mice. G. FIG. @Plin^{+ / +}And plin^{− / −}Weight of isolated gastrocnemius muscle in mice (% of total body weight).
[0047]
3A to 3D.効果 Effect of perilipin inactivation on adipose tissue. A. Histology of adipose tissue. plin^{+ / +}(Left) and plin^{− / −}(Right) Sections of white adipose tissue (WAT, subcutaneous, epididymis) and brown adipose tissue (BAT, interscapular) deposits in mice. Note that there is a difference in magnification between WAT and BAT. B. @Plin^{+ / +}And plin^{− / −}Size distribution of adipocytes in epididymal fat depots in mice. The area of individual adipocytes was measured on sections of epididymal fat. C. @Plin^{+ / +}And plin^{− / −}DNA content of epididymal fat in mice. D. @Plin^{+ / +}And plin^{− / −}Mean size of brown adipocytes in interscapular fat depot in mice.
[0048]
4A-4E. A. @Plin^{+ / +}And plin^{− / −}Effect of 48 h fasting on body weight and plasma parameters in mice. B.ウ ェ ス タ ン Western blotting of HSL extracted from epididymal fat. C. ＨHSL activity of isolated fat cells. HSL activity was determined as described in "Methods"^{+ / +}And plin^{− / −}It was measured in whole cell lysates of subcutaneous and epididymal fat isolated from mice. This activity is expressed in terms of adipose tissue weight (g). D.脂肪 Lipolysis rate in isolated fat cells. Fat cells are plin^{+ / +}And plin^{− / −}It was isolated from mouse epididymal fat. Lipolytic activity was measured in the absence and presence of CL 316,243 (CL) (2 μM) as described in “Methods”. E. FIG. @Plin^{+ / +}And plin^{− / −}Lipolysis in mice. Glycerol, a lipolysis product in plasma, and non-esterified (free) fatty acids (NEFA) were combined with isoproterneol (isopro) (10 mg / kg IP, upper) and CL 316,243 (CL) (0.1 mg / kg IP). , Below) before and after.^*p <0.05;^**p <0.001.
[0049]
5A-5E. A. ＰPlin kept at 21 ℃^{+ / +}And plin^{− / −}Mouse body temperature. Number of animals tested: male plin^{+ / +} 16, plin^{− / −} 12; female plin^{+ / +} 6, plin^{− / −} 11. B.体 Body temperature of mice exposed to 4 ° C. ambient temperature without food. Fasting began when the mice were first exposed to this temperature (n = 6 in both groups, all male mice). C.体 Body temperature of mice exposed to an ambient temperature of 4 ° C. with food (n = 5 in both groups, all male mice).^* plin^{− / −}Is plin^{+ / +}Significantly different from p <0.005. D. $ Plin fed diet containing 35% fat^{+ / +}And plin^{− / −}Representative oxygen consumption (VO) from indirect calorimetry in mice₂) Tracing. VO₂Peaks correspond to active periods, and baseline represents resting values. E. FIG. １４ 14 week old db / db / plin fed normal diet^{+ / +}And db / db / plin^{− / −}Representative VO in mice₂Tracing.
[0050]
6A-6D. A. Ｐ plin after 3.5 months of regular diet or 35% fat (HF) diet^{+ / +}And plin^{− / −}Epididymal fat pad amount of a group of mice (n = 5 per group of male mice). The data before feeding the HF diet was the same as shown in FIG. 2C. B. Ｐ plin after 3.5 months of regular diet or 35% fat (HF) diet^{+ / +}And plin^{− / −}Total carcass fat content of mice (n = 5 per male mouse, group). The data before feeding the HF diet was the same as shown in FIG. 2D. C. Db / db / plin^{+ / +}, Db / db / plin^{− / −}, Plin^{+ / +}And plin^{− / −}Photo of mouse. D.磁 気 Magnetic resonance imaging (MRI) of body fat distribution. Above, plin^{+ / +}(Left panel) and plin^{− / −}(Right panel) MRI of mouse. Lower, db / db / plin^{+ / +}(Left panel) and db / db / plin^{− / −}(Right panel) MRI of mouse. The four in vivo MRI represent cross-sections from the abdominal region of a representative mouse of each different genotype. Spin echo MRI was performed with a repetition time of 2.5 seconds, echo time of 12 ms, field of view 6-x6-cm, slice 1.5-mm, and multi-slices obtained using 128- or 256-phase encoding steps. Was selected from the set. Respiratory gating was applied to reduce motion artifacts. The proton NMR spectrum shown above each image represents the water and lipid signals from the whole mouse. This spectrum was obtained by a single pulse acquisition sequence. Each spectrum was integrated in the 6.5-3.5 ppm and 3.5-0.5 ppm regions to estimate water and lipid concentrations for the whole body.
[0051]
7A-7B. A.雄 Male db / db (triangle) over 4-17 weeks, plin^{+ / +}(Squares), and db / db / plin^{− / −}(Circle) Mouse weight. B.雌 Female db / db (triangles), plin over 4-22 weeks^{+ / +}(Squares), and db / db / plin^{− / −}(Circle) Mouse weight.
[0052]
8A-8B. A.雄 Male db / db (squares), plin over 6-27 weeks^{+ / +}(Diamond), and db / db / plin^{− / −}(Triangle) Weight of mouse. B.雌 Female db / db (square), plin over 6-24 weeks^{+ / +}(Diamond), and db / db / plin^{− / −}(Triangle) Weight of mouse.
[0053]
FIG. Β-oxidation step.
[0054]
FIG. Expression of fatty acyl-CoA dehydrogenase. Wild type (plin^{+ / +}) Mouse and plin^{− / −}Fatty acyl-CoA dehydrogenase, which is a β-oxidase in mouse white adipose tissue (WAT), brown adipose tissue (BAT), liver, heart and muscle, especially very long chain fatty acyl-CoA dehydrogenase (VLCAD) and long chains Fat acyl-CoA dehydrogenase (LCAD) mRNA expression (determined by Northern blot analysis).
[0055]
FIG.発 現 Expression of trifunctional proteins. Wild type (plin^{+ / +}) Mouse and plin^{− / −}MRNA expression of thiolase (trifunctional protein β) and LCHAD (trifunctional protein α) in white adipose tissue (WAT), brown adipose tissue (BAT), liver, heart, and muscle of mice (determined by Northern blot analysis) .
[0056]
FIG.発 現 Expression of uncoupling protein (UCP). Wild type (plin^{+ / +}) Mouse and plin^{− / −}UCP-1 and UCP-2 mRNA expression in mouse adipose tissue (WAT), brown adipose tissue (BAT), liver, heart, and muscle (as determined by Northern blot analysis).
[0057]
13A-13B. A.血漿 Fasting plasma glucose levels. Lep after fasting for 10 hours^{+ / +}/ Plin^{+ / +}Mouse, Lep^{+ / +}/ Plin^{− / −}Mouse, Lep^{ob / ob}/ Plin^{+ / +}Mouse and Lep^{ob / ob}/ Plin^{− / −}Glucose concentration in mouse plasma. B.血漿 Fasting plasma insulin levels. Lep after fasting for 10 hours^{+ / +}/ Plin^{+ / +}Mouse, Lep^{+ / +}/ Plin^{− / −}Mouse, Lep^{ob / ob}/ Plin^{+ / +}Mouse and Lep^{ob / ob}/ Plin^{− / −}Insulin concentration in mouse plasma.
[0058]
14A-14B. Glucose intolerance test. Before administering glucose for the glucose intolerance test, Lep^{+ / +}/ Plin^{+ / +}Mouse, Lep^{+ / +}/ Plin^{− / −}Mouse, Lep^{ob / ob}/ Plin^{+ / +}Mouse and Lep^{ob / ob}/ Plin^{− / −}The mice were fasted for 10 hours. A. Lep^{+ / +}/ Plin^{+ / +}Mouse (diamond), Lep^{+ / +}/ Plin^{− / −}Mouse (square), Lep^{ob / ob}/ Plin^{+ / +}Mouse (triangle) and Lep^{ob / ob}/ Plin^{− / −}Plasma glucose concentrations immediately (0 min), 30 min, 60 min and 90 min after intraperitoneal administration of 1.5 g / kg glucose to mice (circles). B. Lep^{+ / +}/ Plin^{+ / +}Mouse (diamond), Lep^{+ / +}/ Plin^{− / −}Mouse (square), Lep^{ob / ob}/ Plin^{+ / +}Mouse (triangle) and Lep^{ob / ob}/ Plin^{− / −}Plasma insulin concentrations immediately (0 min), 30, 60 and 90 minutes after intraperitoneal administration of 1.5 g / kg glucose to mice (circles).
[0059]
5. Disclosure of the invention
The present invention identifies agents that modulate the activity and / or expression of perilipin as a means of identifying agents useful in preventing and / or treating body weight-related disorders and / or inappropriate regulation of lipid metabolism. To perform screening assays. In particular, the present invention provides in vitro and in vivo assays for identifying compounds that modulate the expression and / or activity of one or more perilipin isoforms. The present invention also relates to the expression and / or activity of signaling molecules such as enzymes (eg, phosphatases, kinases or phosphodiesterases) that modulate the expression and / or activity of perilipin, thereby affecting the expression and / or activity of perilipin. In vitro and in vivo assays for identifying compounds that modulate activity are provided.
[0060]
The present invention also provides a pharmaceutical composition comprising a compound that modulates the activity and / or expression of perilipin protein for the prevention and / or treatment of body weight-related disorders and / or inappropriate regulation of lipid metabolism. Is included. In particular, the pharmaceutical composition can be a perilipin agonist or antagonist. The antagonist may block the interaction of activated perilipin with a signaling pathway downstream thereof, inhibit transcription of the perilipin gene, inhibit processing or translation of perilipin mRNA, or inhibit one or more of the perilipin isoforms. By suppressing the post-translational processing of the foam, it can act to compete with and inhibit another perilipin agonist or antagonist. An agonist can act by activating and / or enhancing the natural biological effect of the perilipin signaling pathway or its expression. In one embodiment, the agonist increases phosphorylation of one or more perilipin isoforms.
[0061]
The present invention comprises administering a pharmaceutical composition that modulates the expression and / or activity of perilipin, the prevention and / or treatment of weight disorders in animals, preferably companion animals, livestock and poultry, and more preferably humans. Provide a method. In one embodiment, the animal is administered an effective amount of a pharmaceutical composition comprising one or more compounds that enhances the weight and performance of the animal by modulating the expression and / or activity of perilipin. In another embodiment, an effective amount of a pharmaceutical composition comprising one or more compounds that reduces body weight by modulating the expression and / or activity of perilipin and ameliorates a condition associated with obesity, Administer to animals in need. In another embodiment, an effective amount of a compound that increases lipid metabolism and increases muscle mass in an animal by modulating the expression and / or activity of perilipin is administered to an animal in need thereof. In another embodiment, an effective amount of a pharmaceutical composition comprising one or more compounds that enhances lipid accumulation in an animal by modulating the expression and / or activity of perilipin is administered to an animal in need thereof. . In yet another embodiment, an effective amount of a pharmaceutical composition comprising one or more compounds that modulates the expression and / or activity of perilipin, thereby improving or delaying the onset or progression of diabetes, is required. Administer to animals.
[0062]
The invention also provides a method of detecting, diagnosing or monitoring the occurrence or progression of diseases and disorders characterized by abnormal expression and / or activity of one or more perilipin isoforms, such as lipid metabolism disorders, weight disorders and diabetes. Also provide. In particular, the present invention provides methods for diagnosing or detecting obesity in an animal by detecting the level of expression and / or activity of perilipin.
[0063]
5.1. Screening assays to identify compounds that modulate the activity and / or expression of perilipin
The invention relates to binding to or expression of perilipin and / or
Provide a method for identifying agents (eg, candidate or test compounds) that have a promoting or inhibiting effect on activity. In one particular embodiment, a method is provided for identifying an agent that modulates phosphorylation of one or more perilipin isoforms, thereby affecting the activity of one or more perilipin isoforms. The invention also provides a method for identifying an agent that modulates the expression and / or activity of an enzyme, such as a phosphatase or kinase, that is involved in regulating the phosphorylation state of one or more perilipin isoforms. Examples of drugs, compounds, candidate compounds or test compounds include, but are not limited to, nucleic acids (eg, DNA and RNA), carbohydrates, lipids, proteins, peptides (including cyclic peptides), peptidomimetics, antibodies, antibody fragments , Small molecules and other substances. Small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, and organic or molecular weights up to about 10,000 g / mole. Inorganic compounds (ie, including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 g / mole, organic or inorganic compounds having a molecular weight of less than about 1,000 g / mole, molecular weight of about 500 Includes organic or inorganic compounds up to g / mole, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. In one particular embodiment, the agent is a small molecule.
[0064]
Compounds that can be tested and identified in the methods described herein include, but are not limited to, compounds obtained from any of the following sources: Aldrich (1001 West St. Paul Ave) , Milwaukee, WI 53233), Sigma Chemical (PO Box 14508, St. Louis, MO 63178), Fluka Chemie AG (Industristrasche 25, CH- 9471 Fuchsche scheuchsche schüzche Buchsche scheuchsche schülche schüsche scheuchscheich). (980 South 2^nd Street, Ronkonoma, NY 11779), Eastman Chemical Company, Fine Chemicals (PO Box 431, Kingsport, TN 37662), and Boehringer Mannheim, Germany, Germany. Using the methods of the present invention, any type of natural product can be screened, including microbial, fungal, plant and animal extracts.
[0065]
Compounds that can be tested for their ability to modulate perilipin expression and / or activity include, but are not limited to, agents that modulate perilipin phosphorylation, and agents and proteins that modulate PKA activity There are agents that modulate phosphatase 1 (PP1) activity. Examples of such agents include, but are not limited to, β-adrenergic agonists (eg, isoproterenol) and phosphodiesterase inhibitors (eg, theophylline, isobutylmethylxanthine, and papaverine). In some embodiments, the compound to be tested does not include an agent that modulates PKA activity, an agent that modulates protein phosphatase 1 (PP1) activity, or an agent that modulates phosphodiesterase activity. In some other embodiments, the agent tested for the ability to modulate perilipin expression and / or activity does not include isoproterenol, theophylline, isobutylmethylxanthine, or papaverine. In some other embodiments, the agent to be tested for its ability to modulate perilipin expression and / or activity includes a weight disorder, a disorder characterized by inappropriate lipid metabolism, a disorder characterized by lipid accumulation, or It does not include compounds that are known or have already been used in the prevention, treatment or amelioration of one or more signs or symptoms associated with diabetes.
[0066]
In addition, a diverse library of drugs, including small molecules, is available. Such libraries can be obtained, for example, from the following sources: Specs and BioSpecs B.A. V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, CA), Contract Services Company (Dolgoprudgy, Moscow Region, USA). (Princeton, NJ), Maybridge Chemicals Ltd. (Cornwall PL24 OHW, United Kingdom) and Asinex (Moscow, Russia).
[0067]
Still further, drugs can be obtained using any of the vast array of combinatorial library methods known in the art, including: biological libraries; spatially addressable parallelism Solid or liquid phase libraries; synthetic libraries requiring deconvolution; "one bead one compound" library method; and synthetic library methods using affinity chromatography selection. Biological library techniques are limited to peptide libraries, while the other four techniques are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145; US Patent No. And U.S. Patent No. 5,807,683, each of which is hereby incorporated by reference in its entirety).
[0068]
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in the following literature: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al., 1944, J. Am. Med. Chem. 37: 2678; Cho et al., 1993, Science 261: 1303; Carrell et al., 1944, Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al., 1944, Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al., 1994, J. Am. Med. Chem. 37: 1233, each of which is incorporated herein by reference in its entirety.
[0069]
A library of compounds can be provided, for example, in the following conditions: in solution (eg, Houghten, 1992, Bio / Techniques 13: 412-421), or on beads (Lam, 1991, Nature 354: 82-84). On chips (Fodor, 1993, Nature 364: 555-556), on bacteria (US Pat. No. 5,223,409), on spores (Patent Nos. 5,571,698; 5,403,484; and). No. 5,223,409), on a plasmid (Cul et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869), or on phage (Scott and Smith, 1990, Science 249: 386-390; Devlin, 1990, Science 249: 404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382; and Felici, 1991, J. Mol. Biol. 222: 301-310). Each of these is incorporated herein by reference in its entirety.
[0070]
5.1.1. Assay for drugs that interact with perilipin
In one embodiment, an agent (ie, a candidate compound) that interacts with (ie, binds to) perilipin (ie, one or more perilipin isoforms) or a fragment thereof (eg, a functionally active fragment) or a perilipin fusion protein is added to a cell. Identify in the based assay system. According to this embodiment, a candidate compound or control compound is contacted with cells expressing perilipin, a fragment thereof, or a perilipin fusion protein, and the ability of the candidate compound to interact with perilipin, a fragment thereof, or a perilipin fusion protein is determined. If desired, the assay can be used to screen a plurality of candidate compounds (eg, a library).
[0071]
The cells used in these assays can be, for example, of prokaryotic (eg, E. coli) or eukaryotic (eg, yeast or mammalian) origin. The cell may be one that can express one or more perilipin isoforms endogenously (eg, steroidogenic cells and adipocytes), or may be a gene that expresses one or more perilipin isoforms or fragments thereof or a perilipin fusion protein. Can be operated. Primary cells or cell lines can be used in the screening assays of the invention. In addition, cells can be obtained from recombinant, transgenic cell lines. For example, cells can be obtained from db / db or ob / ob mice and transformed into a continuous cell line. Examples of techniques that can be used to derive continuous cell lines from transgenic animals are known to those of skill in the art, but are described, for example, in Small et al., 1985, Mol. Cell Biol. 5: 642-648.
[0072]
In a specific example, perilipin (ie, one or more perilipin isoforms) or a fragment thereof or a perilipin fusion protein is labeled so that the binding of the candidate compound to the perilipin, perilipin fragment or perilipin fusion protein is labeled in the complex. The determination can be made by detecting the compound. In another specific example, the candidate compound is labeled to detect binding of the candidate compound to perilipin (ie, one or more perilipin isoforms), a perilipin fragment, or a perilipin fusion protein, and to detect the labeled candidate compound in the complex. By doing so, it can be determined. Without limitation, examples of labels include radioactive labels (³²P,³⁵S or¹²⁵I), fluorescent labels (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine), and enzyme labels (such as horseradish peroxidase, alkaline phosphatase, or luciferase). The ability of a candidate compound to interact directly or indirectly with perilipin, fragments thereof, perilipin fusion proteins can be determined by methods known to those skilled in the art. For example, the interaction between a candidate compound and perilipin, a fragment thereof, or a perilipin fusion protein can be determined by flow cytometry, scintillation assays, immunoprecipitation, or Western blot analysis.
[0073]
In another embodiment, agents that interact with (ie, bind to) perilipin (ie, one or more perilipin isoforms) or a fragment thereof or a perilipin fusion protein are identified in a cell-free assay system. According to this embodiment, a natural or recombinant perilipin (ie, one or more perilipin isoforms), a fragment or perilipin fusion protein thereof, is contacted with a candidate compound or a control compound, and the candidate compound is perilipin, a fragment thereof or a perilipin fusion. Determine the ability to interact with the protein. If desired, the assay can be used to screen a plurality of candidate compounds (eg, a library). Either perilipin or the candidate / control compound can be immobilized to facilitate separation of the conjugated perilipin from its uncomplexed form, as well as to accommodate automation of the assay. Preferably, perilipin or a fragment thereof or perilipin fusion protein is first immobilized. This involves, for example, contacting perilipin or a fragment thereof or a perilipin fusion protein with an immobilized antibody that specifically recognizes and binds it, or binds the protein to a purified preparation of perilipin or a fragment thereof or a perilipin fusion protein. By contacting one surface designed as above. Using techniques known to those skilled in the art, perilipin or a candidate compound can be immobilized on any suitable container (eg, microtiter plates, test tubes, and microcentrifuge tubes). The perilipin or fragment thereof or the perilipin fusion protein may be partially or completely purified (eg, partially or completely free of other polypeptides) or may be part of a cell lysate. The ability of a candidate compound to interact with perilipin or a fragment thereof or a perilipin fusion protein can be determined by methods known to those skilled in the art.
[0074]
In another embodiment, agents that competitively interact (ie, bind) with perilipin (ie, one or more perilipin isoforms) or fragments thereof or perilipin fusion proteins are identified in a competitive binding assay. According to this embodiment, cells expressing perilipin or a fragment thereof or a perilipin fusion protein are contacted with a candidate compound and a compound known to interact with perilipin (eg, protein kinase A), and then the candidate compound is The ability to competitively interact with perilipin or a fragment thereof or a perilipin fusion protein is determined. Alternatively, a candidate compound that competitively interacts (ie, binds) with perilipin or a fragment thereof or a perilipin fusion protein can be combined with a perilipin or a fragment thereof or a perilipin fusion protein and a compound known to interact with the candidate compound and perilipin. Is identified in a cell-free assay system by contacting. As described above, the ability of a candidate compound to competitively interact with perilipin or a fragment thereof or a perilipin fusion protein is determined by methods known to those of skill in the art. These assays can be used to screen multiple candidate compounds (eg, libraries), either cell-based or cell-free.
[0075]
Assays for compounds that interact competitively with perilipin can be performed in a heterogeneous or homogeneous format. In a heterogeneous assay, either perilipin or a fragment thereof, or a perilipin fusion protein, or a control compound known to interact with perilipin (a “binding partner”) is established on a solid phase and solidified at the end of the reaction. Detecting the complex that has settled on the phase. In a homogeneous assay, all reactions are performed in the liquid phase. In either approach, the order of addition of the reactants can be changed to obtain additional information about the compound being tested. For example, by conducting a reaction in the presence of a candidate compound that interferes with the interaction between perilipin and a binding partner, for example, by competition (ie, allowing the candidate compound to precede perilipin and the interactive binding partner). , Or simultaneously with the reaction mixture). Alternatively, a candidate compound that disrupts the formed complex, for example, a compound that has a higher binding constant and displaces one of the components from the complex, is reacted with the candidate compound after the complex is formed. Testing can be done by adding to the mixture. The various schemes are briefly described below.
[0076]
In a heterogeneous assay system, either perilipin or a fragment thereof or a perilipin fusion protein, or a binding partner, is anchored to the surface of the solid phase, while those that do not are directly or indirectly labeled. In practice, it is convenient to use a microtiter plate. The substance to be fixed can be immobilized by non-covalent or covalent bonds. Non-covalent immobilization can be achieved by simply coating the solid surface with perilipin or a fragment thereof or a perilipin fusion protein, or a solution of the binding partner, and drying. Alternatively, an antibody specific to the substance to be fixed can be immobilized and used to fix the substance to the solid surface. This surface can be prepared and stored in advance.
[0077]
To perform this assay, the immobilized material partner is exposed to a coating surface with or without the candidate compound. After the reaction is complete, unreacted components are removed (eg, by washing) and any complexes formed should remain immobilized on the solid surface. Detection of complexes immobilized on the solid phase surface can be achieved by a number of methods. If the non-immobilized substance is pre-labeled, detection of the label immobilized on the surface indicates that a complex has been formed. If the non-immobilized substance is not pre-labeled, indirect labeling may be used, for example, with a labeled antibody specific for the substance that is not initially immobilized (this antibody may be directly labeled or labeled with a labeled anti-Ig antibody). (Indirect labeling is also possible) can be used to detect complexes anchored on the surface. Depending on the order of addition of the reaction components, candidate compounds that inhibit complex formation or destroy the complex formed can be detected.
[0078]
Alternatively, the reaction can be performed in the liquid phase in the presence or absence of the candidate compound, separating the reaction product from unreacted components and detecting the complex. This includes, for example, an immobilized antibody specific for one of the binding components to fix the complex formed in solution, and a labeled antibody specific to the other partner to detect the fixed complex. By using. Again, depending on the order of addition of the reactants to the liquid phase, candidate compounds that inhibit the complex or destroy the formed complex can be identified.
[0079]
In another embodiment of the present invention, a homogeneous assay can be used. In this method, perilipin, a fragment thereof or a perilipin fusion protein, or a binding partner is labeled, and is prepared by forming a complex of perilipin, a fragment thereof, or a perilipin fusion protein and a binding partner in advance, The signal resulting from the label is eliminated by complex formation (see, for example, Rubenstein, US Pat. No. 4,109,496, which utilizes this technique for immunoassays). Addition of a candidate compound that competes with and displaces one of the substances in the preformed complex should generate a signal above background. By this method, candidate compounds that disrupt the perilipin, fragment thereof or perilipin fusion protein / binding partner interaction can be identified.
[0080]
In another embodiment, perilipin (ie, one or more perilipin isoforms) or a fragment thereof is used as a “bait protein” in a two-hybrid assay or a three-hybrid assay to provide another perilipin that binds or interacts with perilipin. Proteins can be identified (eg, US Pat. No. 5,283,317; Zervos et al., 1993, Cell 72: 223-232; Madura et al., 1993, J. Biol. Chem. 268: 12046-12054; Bartel). Et al., 1993, Bio / Techniques 14: 920-924; Iwabuchi et al., 1993, Oncogene 8: 1693-1696; and PCT Publication No. WO 94/10300). One of skill in the art will appreciate that such binding proteins may also be involved in the transmission of signals by perilipin, for example, as an upstream or downstream element of a signaling pathway involving perilipin.
[0081]
5.1.2. Assays for drugs that modulate perilipin expression or activity
In another embodiment, an agent (ie, a candidate compound) that modulates the expression of perilipin (ie, one or more perilipin isoforms) is treated with a cell (eg, a prokaryote) that expresses perilipin (ie, one or more perilipin isoforms). The cells are identified by contacting a candidate or control compound (eg, phosphate buffered saline (PBS)) with cells of origin or eukaryotic origin and determining the expression of perilipin or mRNA encoding perilipin. The level of expression of perilipin or mRNA encoding perilipin in the presence of the candidate compound is compared to the level of expression of perilipin or mRNA encoding perilipin in the absence of the candidate compound (eg, in the presence of a control compound). The candidate compound is then identified as a modulator of perilipin expression based on this comparison. For example, if the expression of perilipin or mRNA encoding perilipin is significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as an enhancer of expression of perilipin or mRNA encoding perilipin. Alternatively, if the expression of perilipin or mRNA encoding perilipin is significantly less in the presence of the candidate compound than in the absence thereof, the candidate compound is identified as an inhibitor of the expression of perilipin or mRNA encoding perilipin. The level of expression of perilipin or mRNA encoding perilipin can be measured by methods known to those skilled in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR, and protein levels can be assessed by Western blot analysis and immunoassays such as ELISA. In certain embodiments of the invention, the expression of perilipin A, perilipin B, perilipin C, or a combination thereof is measured.
[0082]
In another embodiment, the agent that modulates the activity of perilipin (ie, one or more perilipin isoforms) is a preparation containing perilipin or a cell that expresses perilipin (ie, one or more perilipin isoforms) (eg, A prokaryotic or eukaryotic cell is contacted with a candidate or control compound and identified by determining the ability of the candidate compound to modulate (eg, enhance or suppress) the activity of perilipin. Perilipin activity can be assessed by the following detections: detection of perilipin phosphorylation; perilipin cellular signaling pathways (eg, intracellular Ca²⁺, Diacylglycerol, IP3, etc.); detection of the activity of an enzyme whose activity is regulated by perilipin (eg, hormone-sensitive lipase activity); reporter gene (eg, perilipin-responsive, one of the detectable markers, eg, Detection of the induction of a regulatory element operably linked to a luciferase-encoding nucleic acid; or a cellular response, such as lipid hydrolysis (eg, O₂Detection of changes in consumption or release of glycerol from adipocytes) or detection of the distribution of lipid droplets. Techniques known to those of skill in the art and those described herein can be used to measure these activities (eg, Holmes et al., 1997, Methods Enzymol. 286: 45-67 and Martinez- See Botas et al., 2000, Nature Genetics 26: 474-479, each of which is incorporated herein by reference). The candidate compound can then be identified as a modulator of perilipin activity by comparing the effect of the candidate compound to a control compound. Suitable control compounds include phosphate buffered saline (PBS) and standard saline (NS).
[0083]
In yet another embodiment, agents that modulate the expression, activity, or both expression and activity of perilipin (ie, one or more perilipin isoforms) are identified in an animal, preferably a mammal. Examples of suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. Preferably, the animals used are the following obesity models: leptin resistant animals (eg db / db mice), melanocortin-4 receptor knockout mice (MR-4)^{− / −}), Leptin-deficient mice (ob / ob mice), tubby mice (tubby protein deficient), fa / fa (Zucker Diabetic Fatty or ZDF) rats, melanocortin-3 receptor knockout mice, POMC deficient mice, and fat / Fat mice (see, e.g., Barsh et al., 2000, Nature 404: 644-651; Fisher et al., 1999, Int. J. Obes. Rel. Metab. Disord. 23 Suppl: 54-58; Giridhran, 1998). , Indian J. Med.Res. 108: 225-242; Zhang et al., 1994, Nature 372: 425-432; Noben-Trauth et al., 1996, Nature 3. 80: 534-538; Iida et al., 1996, BBRC 224: 597-604; Phillips et al., 1996, Nature Genetics 13: 18-19; Chen et al., 2000, Nature Genetics 26: 97-102; Butler et al., 2000, Endo. 141: 3518-3521; Yawen et al., 1999, Nature Medicine 5: 1066-1070; and Naggert et al., 1995, Nature Genetics 10: 135-142). According to this embodiment, the candidate or control compound is administered to a suitable animal (eg, orally, rectally or subcutaneously, intramuscularly, intraperitoneally or intravenously, such as intravenously), and expression, activity or expression of perilipin is determined. The effect on both activities is determined. A candidate compound that alters expression of perilipin encodes perilipin or perilipin in an animal or group of animals treated with a control compound, the level of mRNA encoding perilipin or perilipin in an animal or group of animals treated with the candidate compound. Can be identified by comparing to the level of the mRNA. Techniques known to those of skill in the art can be used to determine mRNA and protein levels, for example, in situ hybridization. Candidate compounds that alter the activity of perilipin can be identified by assaying for perilipin activity in animals treated with the control compound and in animals treated with the candidate compound. Perilipin activity can be assessed by the following detections: detection of perilipin phosphorylation; perilipin cellular second messenger (eg, intracellular Ca²⁺, Diacylglycerol, IP3, etc.); detection of reporter gene induction; or cellular response (eg, distribution of perilipin on lipid droplets, or O2).₂Detection of lipid metabolism, such as lipid hydrolysis, by detecting changes in consumption or changes in glycerol release from adipocytes. To detect a change in the activity of perilipin, techniques known to those of skill in the art and described herein can be used.
[0084]
5.1.3. Computer modeling compound
Computer modeling and search techniques allow the identification of compounds that can modulate perilipin gene expression and / or activity, or the improvement of previously identified compounds. After identifying such a compound or composition, it is preferable to identify the active site or region. The active site is identified using methods known in the art, for example, studying the amino acid sequence of a peptide, the nucleotide sequence of a nucleic acid, or the complex of a related compound or composition with a natural binding partner. be able to. For example, the active site can be found by finding where the complexed binding partner is present in the agent using chemical methods or X-ray crystallography.
[0085]
Next, it is preferable to determine the geometric three-dimensional structure of the active site. This can be performed by known methods, such as X-ray crystallography, which can determine the complete molecular structure. Also, solid or liquid phase NMR can be used to determine a given intramolecular distance within an active site and / or in a complex with a binding partner. Any other experimental method of structure determination can be used to obtain a partial or complete geometric structure. The geometric structure can also be measured using natural or artificial complexed ligands, which can increase the accuracy of the determined active site structure.
[0086]
Computer-based methods of numerical modeling can be used to complete or improve the accuracy of the structure (eg, in embodiments where an incomplete or poorly accurate structure has been determined). Any of the modeling techniques recognized in the art can be used. These include, but are not limited to, parameterized models specific to particular biopolymers, such as proteins or nucleic acids, molecular dynamics models based on computational calculations of molecular motion, statistical mechanical models based on thermal ensembles , Or a composite model. For most types of models, standard intermolecular force fields representing the forces between constituent atoms and constituent groups are required and can be selected from force fields known in physical chemistry. Non-limiting exemplary force fields known in the art and that can be used in such models include the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field. included. Incomplete or inaccurate experimental structures can serve as constraints on the complete and more accurate structures calculated by these modeling methods.
[0087]
Finally, after determining the structure of the active site, either experimentally, by modeling, or a combination thereof, the candidate modulator compound is identified by searching a database containing information about the molecular structure of the compound along with the compound. Can be identified. Such searches search for compounds that have a structure that matches the determined active site structure and that interacts with the groups that define the active site. Such searches may be manual, but are preferably computer assisted. The compounds found in this search are those that modulate a potential target or pathway gene product.
[0088]
Alternatively, these methods can be used to identify modulator compounds that are already known or improved from binding partners. The composition of a known compound can be modified and the new composition can be used to apply the experimental and computer modeling methods described above to determine the structural effects of the modification. The altered structure is then compared to the compound's active site structure to determine if an improved fit or interaction results. In this way, a modified modulator compound or binding partner with improved specificity or activity is obtained by rapidly assessing systematic changes in composition, such as by changing side-chain groups.
[0089]
Other experimental and computer modeling methods useful for the identification of modulator compounds based on the identification of the active site of a target or pathway gene or gene product and associated transduction and transcription factors will be apparent to those skilled in the art.
[0090]
Examples of molecular modeling systems are the CHARMm and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMm performs energy minimization and molecular dynamics functions. QUANTA performs the construction, schematic modeling and analysis of molecular structures. QUANTA allows the interaction structure, modification, visualization and analysis of molecular behavior.
[0091]
Numerous literature reviews computer modeling of drugs that interact with specific proteins: Rotivinen et al., 1988, Acta Pharmaceutical Fennica 97: 159-166; Ripka, (June 16, 1988), New Scientist 54-57; McKinally and Rossmann, 1989, Annu. Rev .. Pharmacol. Toxiol. 29: 111-122; Perry and Davies, OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. London. 236: 125-140 and 1-162; and for model receptors for nucleic acid components, see Askew et al., 1989, J. Am. Am. Chem. Soc. 111: 1082-1090. Other computer programs that screen chemicals and are shown graphically are available from BioDesign, Inc. (Pasadena, CA.), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these were originally designed to apply drugs specific to specific proteins, after DNA or RNA regions were identified, they were designed to design drugs specific to these regions. Can be applied.
[0092]
Although described generally above with reference to the design and production of compounds that can alter binding, for compounds that become inhibitors or activators, natural or synthetic chemicals, and biologically active substances such as proteins It is also possible to screen libraries of known compounds containing
[0093]
5.2. Perilipin antagonist
Compounds identified by the assays described in Section 5.1 above that antagonize perilipin (ie, one or more perilipin isoforms) by reducing or suppressing perilipin expression and / or activity levels. It can be used in accordance with the present invention to prevent, treat or ameliorate one or more signs or symptoms associated with obesity. Moreover, such compounds can be used to prevent, treat or ameliorate one or more signs or symptoms associated with diabetes. In addition, such compounds are used to prevent, treat or ameliorate one or more signs or symptoms associated with lipid metabolism disorders (eg, lipodystrophy) or disorders characterized by lipid accumulation (eg, atherosclerosis). can do. Furthermore, such compounds can be used to increase lipid metabolism and increase muscle mass. As discussed in Section 5.1 above, such compounds include, but are not limited to, nucleic acids (eg, antisense nucleic acids and triple-stranded helix molecules), peptides, phosphopeptides, small organic or inorganic molecules, or antibodies ( For example, polyclonal, monoclonal, human, humanized, anti-idiotype, chimeric or single chain antibodies, as well as Fab, F (ab ')₂And Fab expression library fragments, as well as their epitope binding fragments. In certain embodiments, the agent that suppresses or reduces perilipin expression and / or activity is one or more fragments of perilipin isoforms or derivatives thereof, or ones that endogenously prevent perilipin from functioning properly. A fusion protein containing the above-described fragment of perilipin isoform or a derivative thereof.
[0094]
5.2.1. Perilipin nucleic acid antagonists and pharmaceutical compositions based thereon
The present invention provides perilipin antisense nucleic acids, ribozymes, and triple-stranded helical molecules that target perilipin expression. The invention further provides a pharmaceutical composition comprising one or more perilipin antisense nucleic acids, triple helical molecules, and / or ribozymes and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be used for the prevention or treatment of obesity and disorders associated with impaired or incapable lipid metabolism. Further, such pharmaceutical compositions can be used for the prevention or treatment of diabetes (eg, diabetes mellitus). Still further, such pharmaceutical compositions can be used to increase lipid metabolism and muscle mass in animals, preferably livestock and poultry, more preferably humans.
[0095]
5.2.1.1. Perilipin antisense nucleic acid
In certain embodiments, perilipin expression is suppressed by use of a perilipin antisense nucleic acid. The present invention provides the use of a nucleic acid comprising at least 6 nucleotides that is antisense to a gene or cDNA encoding perilipin or a fragment thereof for treatment or prevention. As used herein, perilipin "antisense" nucleic acid refers to a nucleic acid that can hybridize by sequence complementarity with a portion of the RNA (preferably mRNA) encoding perilipin. The antisense nucleic acid need only be complementary to the coding and / or non-coding regions of the mRNA encoding perilipin. In a specific embodiment, the perilipin antisense nucleic acid suppresses or reduces expression of one or more perilipin isoforms. In a preferred embodiment, the perilipin antisense nucleic acid suppresses or reduces perilipin A expression. Such antisense nucleic acids have utility as compounds that suppress perilipin expression and prevent or treat obesity, lipid metabolism disorders (eg, lipodystrophy) or disorders characterized by lipid deposition (eg, atherosclerosis). And to increase lipid metabolism and increase muscle mass.
[0096]
Representative, non-limiting examples of perilipin antisense molecules include:
5'AGGTGAGGCCTTTGTTGACTGCCAT3 '(SEQ ID NO: 1)
5'CTGCTCAGGGGAGTTCCCATCCAG3 '(SEQ ID NO: 2)
[0097]
A perilipin antisense nucleic acid is an oligonucleotide of at least 6 nucleotides, preferably an oligonucleotide ranging from 6 to about 50 nucleotides. In certain embodiments, the perilipin antisense nucleic acid is an oligonucleotide of at least 10, at least 15, at least 25, at least 50, at least 75, at least 100, at least 150, or at least 200 nucleotides. The perilipin antisense nucleic acid may be DNA or RNA or a chimeric mixture or derivative or variant thereof, and may be single-stranded or double-stranded. Perilipin antisense nucleic acids can be modified with a base moiety, a sugar moiety, or a phosphate backbone. Perilipin antisense nucleic acids may contain other accessory groups such as: peptides; cell membranes (eg Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or the blood-brain barrier (eg, PCT Publication No. WO 89/10134, 1988). See, published April 25, 1989) agents that enhance transport across cross-linking agents; and cleavage agents that trigger hybridization (see, eg, Kroll et al., 1988, BioTechniques 6: 958-976) or intercalating agents (eg, Zon, 1988, Pharm. Res. 5: 539-549).
[0098]
In a preferred embodiment of the present invention, the perilipin antisense nucleic acid is a single-stranded DNA. The oligonucleotide may be modified at any position on its structure with substituents generally known in the art.
[0099]
The perilipin antisense nucleic acid may contain one or more of the following modified base components: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl Cytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosyl eosin, inosine, N6-isopentenyl adenine, 1 -Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyl Uracil, 5-meth Cyaminomethyl-2-thiouracil, beta-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine ), Pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, and other base analogs.
[0100]
In another embodiment, the perilipin antisense nucleic acid may include a modified sugar component, for example, one or more of the following sugar components: arabinose, 2-fluoroarabinose, xylylose, and hexose.
[0101]
In another embodiment, a perilipin antisense nucleic acid may include one or more of the following modified phosphate backbones: phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphordiadiate Midate, methyl phosphonate, alkyl phosphotriester, formacetal, or analogs of formacetal.
[0102]
In yet another embodiment, the perilipin antisense nucleic acid is an alpha anomeric oligonucleotide. Alpha anomeric oligonucleotides form specific double-stranded hybrids with complementary RNAs, where the strands are parallel to each other, unlike normal β units (Gautier et al., 1987, Nucl. Acids Res. 15). : 6625-6641).
[0103]
The perilipin antisense nucleic acid may be conjugated to another molecule, eg, a peptide, a hybridization triggered cross-linking agent, a transport agent, or a hybridization triggered cleavage agent.
[0104]
A nucleic acid of the invention, such as a perilipin antisense nucleic acid, is synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (e.g., available from Biosearch, Applied Biosystems, etc.). May be. As an example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16: 3209), and methylphosphonate oligonucleotides can be synthesized using controlled microporous glass polymer supports (Sarin 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451).
[0105]
In certain embodiments, perilipin antisense nucleic acids can be administered directly to cells using techniques known to those of skill in the art, such as, for example, microinjection, electroporation, lipofection, and calcium phosphate precipitation. In another specific embodiment, a perilipin antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, the vector is introduced in vivo so as to be taken up by a cell, and the vector or a part thereof is transcribed in the cell to produce the antisense nucleic acid (RNA) of the present invention. Such a vector may include a sequence encoding a perilipin antisense nucleic acid. Such vectors may remain episomal or may integrate into a chromosome (ie, become part of a chromosome) provided that they can be transcribed to produce the desired antisense RNA. The vector can be constructed by recombinant DNA techniques standard in the art. Vectors may be plasmids, viruses, or others used for replication and expression in mammalian cells. Expression of the sequence encoding perilipin antisense RNA may be from any promoter known in the art to operate in animal cells, preferably mammalian cells, and more preferably human cells. Such a promoter can be inducible, constitutive, or tissue-specific.
[0106]
Examples of promoters that can be used to regulate the expression of perilipin antisense include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304). -310), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 7887-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445), regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors, such as the beta-lactamase promoter. -(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731) or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25); See also "Useful Proteins from Recombinant Bacteria" in Scientific American, 1980, 242: 74-94; nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303: 209-213) or cauliflower mosaic virus. A plant expression vector containing the 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871), and the photosynthetic enzyme ribulose diphosphate Boxylase promoter (Herrera-Estrella et al., 1984, Nature 310: 115-120); from yeast or other fungi such as the Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter. Promoter element. Also included are animal transcription regulatory regions that have been used in transgenic animals with the following tissue specificities: elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639). Ornitz et al., 1986, Cold Spring Harbor Symp.Quant.Biol.50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); an insulin gene regulatory region active in pancreatic beta cells (Hanahan, 1985). , Nature 315: 115-122); Immunoglobulin gene regulatory regions active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature). Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444); a mouse mammary tumor virus control region active in testis, breast, lymphoid and mast cells (Leder et al., 1986, Cell). 45: 485-495); an albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Level. 1: 268-276); an alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol) 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); an α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Level. : 161-171); β-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); Oligodendrocytes in the brain Myelin basic protein gene regulatory region active in skeletal muscle (Readhead et al., 1987, Cell 48: 703-712); myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314: 283-286) ); And a gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378). In certain embodiments, perilipin antisense expression is regulated by a regulatory element that is active or predominantly active in steroidogenic or adipocytes (eg, the leptin promoter).
[0107]
The perilipin antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of the RNA transcript of the gene encoding perilipin, preferably the human gene encoding perilipin. However, although absolute complementarity is preferred, it is not required. As used herein, a sequence that is "complementary to at least a portion of an RNA" is one that has sufficient complementarity to hybridize with the RNA under stringent conditions to form a stable duplex. Means an array that has In the case of double-stranded perilipin antisense nucleic acids, one may therefore test one strand of double-stranded DNA or assay for triple-strand formation. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the hybridizing nucleic acid, the more it will form a stable double strand (or triplex in some cases), even though it contains more base mismatches with the RNA encoding perilipin. One skilled in the art can ascertain an acceptable degree of mismatch by using standard procedures to determine the melting point of the hybridized complex.
[0108]
The present invention provides a pharmaceutical composition comprising an effective amount of a perilipin antisense nucleic acid of the invention and a pharmaceutically acceptable carrier as described below. In certain embodiments, a pharmaceutical composition comprising one or more perilipin antisense nucleic acids is administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, such compositions can be used to achieve sustained release of perilipin antisense nucleic acids.
[0109]
In another embodiment, the present invention relates to a perilipin nucleic acid in a eukaryotic cell (eg, a steroidogenic cell or an adipocyte) comprising providing the cell with an effective amount of a composition comprising a perilipin antisense nucleic acid of the present invention. Methods are provided for suppressing expression of a sequence.
[0110]
The perilipin antisense nucleic acids can be used to prevent or treat diseases and disorders characterized by abnormal perilipin expression, such as lipid metabolism disorders, weight disorders (eg, obesity), and diabetes. In addition, perilipin antisense nucleic acids can be used to increase lipid metabolism and muscle mass. In a preferred embodiment, a single-stranded DNA antisense perilipin oligonucleotide is used.
[0111]
A subject determined to have an effective amount of one or more perilipin antisense nucleic acids in a pharmaceutically acceptable carrier in a pharmaceutically acceptable carrier, the subject having lipid metabolism disorders and obesity or other body weight disorders. Can be administered. A pharmaceutical composition of the invention comprising an effective amount of one or more perilipin antisense nucleic acids in a pharmaceutically acceptable carrier is administered to a subject who has or is predisposed to diabetes (eg, diabetes mellitus). be able to. Administering a pharmaceutical composition of the present invention comprising an effective amount of one or more perilipin antisense nucleic acids in a pharmaceutically acceptable carrier to a subject having or having a disorder characterized by lipid accumulation. can do. The amount of one or more perilipin antisense nucleic acids found to be effective in treating or preventing obesity or diabetes can be determined by standard clinical techniques.
[0112]
5.2.1.2. Inhibitory ribozyme and triple helix approaches
In another embodiment, the gene sequence encoding perilipin is used in conjunction with the well-known gene "knockout", ribozyme, or triple-helix method to reduce perilipin gene expression, thereby reducing the level of perilipin activity. It can be reduced to prevent or treat obesity, disorders of lipid metabolism or disorders characterized by lipid accumulation or diabetes, and / or to increase lipid metabolism and muscle mass. In this approach, ribozymes or triple-helix molecules are used to modulate the activity, expression or synthesis of the gene encoding perilipin, thereby preventing obesity, disorders of lipid metabolism or disorders characterized by lipid accumulation, or diabetes. Or treating and / or increasing lipid metabolism and muscle mass. Such molecules can be designed to reduce or suppress the expression of mutated or non-mutated target genes. Techniques for making and using such molecules are well known to those skilled in the art.
[0113]
Ribozymes designed to catalytically cleave the transcript of the gene mRNA encoding perilipin isoform can be used to prevent translation of the target gene mRNA and thus prevent expression of its gene product ( See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247: 1222-1225).
[0114]
Ribozymes are enzymatic RNA molecules that can catalyze specific cleavage of RNA (for a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by cleavage within the nucleotide. The composition of the ribozyme molecule must include one or more sequences that are complementary to the target gene mRNA (ie, one or more perilipin isoforms) and include the well-known catalytic sequences involved in mRNA cleavage. Must be. See, for example, US Pat. No. 5,093,246 for this sequence, which is incorporated herein by reference in its entirety.
[0115]
To destroy the mRNA encoding perilipin, a ribozyme that cleaves the mRNA at the site of the site-specific recognition sequence can be used, but the use of a hammerhead ribozyme is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the mRNA has the following sequence of two bases: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art and is described in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, pages 33 and 48, especially in New York, pages 19 and 28. , Nature, 334, 585-591, which are incorporated herein by reference in their entirety.
[0116]
Preferably, the ribozyme is engineered to increase efficiency and position intracellular accumulation of non-functional mRNA transcripts by positioning the cleavage recognition site near the 5 'end of the mRNA encoding the perilipin isoform. Minimize.
[0117]
The ribozymes of the present invention include RNA endoribonucleases (hereinafter referred to as IVS or L-19 IVS RNA), such as those present in Tetrahymena thermophila, described in detail by Thomas Cech and coworkers. "Cech-type ribozymes") (Zaug et al., 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug et al., 1986, Nature, 324, 429-433; Published International Patent Application No. WO 88/04300 by University Patents Inc .; and Been and Tech, 1986, Cell, 47, 207. 216). Cech-type ribozymes have an eight base pair active site that hybridizes to the target RNA sequence, at which point cleavage of the target RNA occurs. The present invention encompasses these Cech-type ribozymes which target an eight base pair active site sequence present in the gene encoding perilipin.
[0118]
As with the antisense approach, ribozymes can also include modified oligonucleotides (eg, for improved stability, targeting, etc.) and should be delivered to cells expressing perilipin in vivo. A preferred method of delivery is to use a DNA construct that "encodes" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that the transfected cells have sufficient ribozyme. Produces and destroys endogenous mRNAs encoding perilipin and also suppresses translation. Because ribozymes are catalytic, unlike antisense molecules, the intracellular concentrations required for efficacy may be relatively low.
[0119]
Endogenous perilipin expression can also be reduced by inactivating or “knocking out” the gene encoding perilipin or the promoter of such a gene using targeted homologous recombination (eg, Smithies et al., 1985, Nature 317). Thomas and Capecchi, 1987, Cell 51: 503-512; Thompson et al., 1989, Cell 5: 313-321; and Zijlstra et al., 1989, Nature 342: 435-438, all of which are by reference. Incorporated herein). For example, a mutant gene (or a completely unrelated DNA sequence) encoding a non-functional perilipin flanked by DNA homologous to an endogenous gene (either the coding region or the regulatory region of the gene encoding perilipin) Used with or without a selectable marker and / or a negative selectable marker to transfect cells that express the target gene in vivo. Insertion of the DNA construct by targeted homologous recombination results in inactivation of the target gene. Such techniques are particularly suitable in the agricultural field, where ES (embryonic stem) cell modifications have been used to create animal progeny with inactive target genes (eg, Thomas and Capecchi, 1987). And Thompson, 1989, supra). However, it is also possible to apply this technique to humans if the recombinant DNA construct is administered directly to the required site in vivo using an appropriate viral vector, or if that site is targeted.
[0120]
Alternatively, the endogenous expression of the gene encoding perilipin is targeted to a deoxyribonucleotide sequence that is complementary to the regulatory region of the gene (ie, the gene promoter and / or enhancer) to encode perilipin in target cells in the body. It can be reduced by forming a triple-stranded helical structure that interferes with gene transcription (generally, Helene, 1991, Anticancer Drug Des., 6 (6), 569-584; Helene et al., 1992). , Ann.NY Acad.Sci., 660, 27-36; and Maher, 1992, Bioassays 14 (12), 807-815).
[0121]
Nucleic acid molecules used in triple-helix formation for repression of transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple-helix formation according to Hoogsteen base-pairing rules. This generally requires that there be an affordable stretch of purine or pyrimidine on one strand of the duplex. The nucleotide sequence may be pyrimidine-based, which results in TAT and CGC between the three associated strands of the resulting triple helix.⁺Bring the triplet. The pyrimidine-rich molecule provides base complementarity to the purine-rich region of one strand of the duplex in a direction parallel to this strand. In addition, purine-rich (eg, containing a stretch of G residues) nucleic acid molecules can be selected. These molecules form a triple-stranded helix with a GC-pair rich DNA duplex (where most of the purine residues are located on one strand of the target duplex), resulting in a triple-stranded helix. GGC triplets are formed between the three chains.
[0122]
Alternatively, the potential target sequence for the formation of a triple-stranded helix can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3 ', 3'-5' fashion, so that they first base pair with one strand of the duplex and then with the other. And eliminates the need for the presence of affordable stretches of purines or pyrimidines on one strand of the duplex.
[0123]
In cases where the expression of the mutated gene is suppressed using an antisense, ribozyme or triple-helix molecule as described herein, the technique involves transcription of mRNA produced by the normal allele of perilipin (triplicate). It is believed that the concentration of perilipin present will be lower than that required for a normal phenotype, as it will very effectively reduce or suppress strand helix) or translation (antisense, ribozyme). In such cases, nucleic acid encoding and expressing perilipin exhibiting normal gene activity using gene therapy to ensure that the level of activity of the gene encoding perilipin is maintained substantially normal Sequences (but nucleic acid molecules that do not contain sequences susceptible to any antisense, ribozyme or triple-helix therapy, if used) can be introduced into cells. The nucleic acid sequence encoding perilipin is obtained, for example, from the GenBank database (eg, see GenBank Accession No. AB005293 for nucleic acid sequences encoding human perilipin) or similar databases, from publications, or by conventional cloning and sequencing. be able to. Alternatively, if the gene encodes an extracellular protein, normal perilipin can be co-administered to maintain the required level of perilipin activity.
[0124]
The antisense RNAs and DNAs, ribozymes, and triple-stranded helical molecules of the invention can be prepared by any method known in the art for the synthesis of DNAs and RNAs as described above. These include techniques for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be made by in vitro and in vivo transcription of a DNA sequence encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate a suitable RNA polymerase promoter, such as a T7 or SP6 polymerase promoter. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be stably introduced into cell lines.
[0125]
5.3. Perilipin agonists
Compounds that agonize perilipin by increasing perilipin expression and / or activity (i.e., one or more perilipin isoforms), identified by the assay of Section 5.1 above, can be used in accordance with the present invention to: One or more signs or symptoms associated with a disorder characterized by weight loss (eg, cachexia and anorexia) can be prevented, treated, or ameliorated. In addition, perilipin agonists can be used to increase lipid accumulation for weight gain. As discussed in Section 5.1 above, such compounds include, but are not limited to, nucleic acids, proteins, peptides, phosphopeptides, small molecules, or antibodies (eg, polyclonal, monoclonal, human, humanized, anti-idiotype). , Chimeric or single chain antibodies, as well as Fab, F (ab ')₂And Fab expression library fragments, as well as their epitope binding fragments.
[0126]
In one particular embodiment, one or more perilipin isoforms or functional fragments thereof are administered in a dose sufficient to increase the activity of perilipin in vivo, eg, by mimicking the function of perilipin in vivo. , Administered to animals. In another specific embodiment, an analog or derivative of a perilipin isoform or functional fragment thereof is sufficient to increase the activity of perilipin in vivo, for example, by mimicking the function of perilipin in vivo. The dose is administered to the animal. In another embodiment, the activity of perilipin is enhanced by mimicking perilipin isoforms, functional fragments of perilipin isoforms, or fusion proteins comprising analogs or derivatives thereof, for example, in vivo, to the function of perilipin. The animal is administered a dose sufficient to increase in vivo.
[0127]
Proteins and peptides that can be used in such methods include synthetic (eg, recombinant or chemically synthesized) proteins and peptides, as well as naturally occurring proteins and peptides. The proteins and peptides may contain naturally occurring and / or non-naturally occurring amino acid residues (eg, D-amino acid residues) and / or one or more non-peptide bonds (eg, imino, ester, hydrazide, semicarbazide and azo bonds). ). The protein or peptide may also contain other chemical groups (eg, functional groups) at the amino and / or carboxy terminus, for example, such that the stability, bioavailability and / or inhibitory activity of the peptide is enhanced. You may go out. Representative functional groups include hydrophobic groups (eg, carbobenzoxyl, dansyl, and t-butyloxycarbonyl), acetyl, 9-fluorenylmethoxycarbonyl, and macromolecular carrier groups (eg, lipid-fatty acids). Conjugates, polyethylene glycol or carbohydrates) and peptide groups. In one embodiment, the proteins and peptides used in such methods have one or more amino acid substitutions, additions or deletions introduced into the encoded protein or peptide. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Alternatively, mutations may be randomly introduced into all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants screened for their biological activity and suddenly retain or antagonize the activity. Mutants can be identified. After mutagenesis, the encoded protein can be recombinantly expressed and the activity of the protein determined.
[0128]
When the compound to be administered is a peptide compound, the DNA sequence encoding the peptide compound can be directly administered to an animal. Any of the techniques discussed below for performing intracellular administration of a compound (eg, liposome administration, etc.) can be used to administer such a DNA molecule. DNA molecules can be produced, for example, by well-known recombinant techniques.
[0129]
If the abnormal perilipin gene is involved in the disorder, the animal can be treated by gene replacement therapy. One or more copies of the normal perilipin gene or a portion of the gene that leads to the production of normal perilipin with normal perilipin gene function can be inserted into cells. In certain embodiments, the nucleic acid sequence is directly administered in vivo, and is expressed so as to produce the encoded product. This can be achieved by any of a number of methods known in the art. For example, they may be constructed as part of a suitable nucleic acid expression vector and administered such that they enter the cell, such as by infection using a defective or attenuated retrovirus or other viral vector (US Pat. 4,980,286); direct injection of naked DNA; microparticle bombardment (eg, a gene gun; Biolistic, Dupont), coating with lipids or cell surface receptors, transfectants, or liposomes, microparticles or microparticles. Use encapsulation within a capsule; or administer linked to a peptide known to enter the nucleus or linked to a ligand that undergoes receptor-mediated endocytosis (eg, Wu and Wu, 1987). , J. Bi . L.Chem 262: 4429-4432 reference) (which can be used to target cell types specifically expressing the receptors); and others. In another embodiment, a nucleic acid-ligand complex is formed, wherein the ligand comprises a fusogenic viral peptide to degrade endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication WO 92/06180 (Apr. 1992). WO 92/22635 (issued December 23, 1992) (Wilson et al.); WO 92/20316 (issued November 26, 1992) (Findeis et al.); WO 93/14188. (Issued July 22, 1993) (Clarke et al.); See WO 93/20221 (issued October 14, 1993) (Young). Alternatively, the nucleic acid can be introduced into a cell, incorporated into host cell DNA by homologous recombination, and expressed (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; And Zijlstra et al., 1989, Nature 342: 435-438).
[0130]
In one embodiment, a viral vector containing a nucleic acid encoding perilipin A, perilipin B, perilipin C, or any combination thereof is used. For example, a retroviral vector can be used. The nucleic acid encoding perilipin for use in gene therapy is cloned into one or more vectors useful for delivering the gene to a patient. Further details on retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6: 291-302, where the mdr1 gene is delivered to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. The use of retroviral vectors has been described. Other references describing the use of retroviruses in gene therapy include: Clowes et al., 1994, J. Am. Clin. Invest. 93: 644-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Level. 3: 110-114.
[0131]
Lentiviruses can also be used for gene therapy. Details on the use of lentiviruses in gene therapy can be found, for example, in the following literature: Evans et al., 1999, Human Gene Therapy 10: 1479-1489; Han et al., 1999, Human Gene Therapy 10: 1867-1873; and Zufferey et al., 1997, Nature Biotechnology 15: 871-875.
[0132]
Adenoviruses are another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503 provide a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5: 3-10, demonstrate the use of adenovirus vectors to transfer genes into the respiratory epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in the following literature: Rosenfeld et al., 1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; Mastranelli et al., 1993. J. Clin. Invest. 91: 225-234; PCT Publication WO 94/12649; Wang et al., 1995, Gene Therapy 2: 775-783; Chan, 1995, Curr. Opin. Lipidol. 6: 335-340; and Oka et al., 2000, Curr. Opin. Lipidol. 11: 179-186. In a preferred embodiment, an adenovirus vector is used to express one or more perilipin isoforms or fragments thereof.
[0133]
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300; and US Pat. No. 5,436,146). .
[0134]
Another approach to gene therapy involves introducing a gene into cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection or viral infection. Usually, the method of introduction involves the introduction of a selectable marker into the cells. The cells are then sorted to isolate those cells that have taken up and are expressing the introduced gene. These cells are then delivered to the patient.
[0135]
In this embodiment, the nucleic acid is introduced into cells, and the resulting recombinant cells are administered in vivo. Such introduction can be performed by any method known in the art. Without limitation, transfection, electroporation, microinjection, infection with a virus or bacteriophage vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. There is. Numerous techniques for introducing foreign genes into cells are known in the art (eg, Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol. 217). : 618-644; and Cine, 1985, Pharmac. Ther. 29: 69-92), which can be used in accordance with the present invention provided that the essential development and physiological function of the recipient cells is not disrupted. . The technique should provide for stable transfer of the nucleic acid into the cell, so that the nucleic acid can be expressed by the cell, and preferably inherited and expressed by progeny cells.
[0136]
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Preferably, recombinant blood cells (eg, hematopoietic stem or progenitor cells) are administered intravenously. The amount of cells intended for use will depend on the desired effect, patient condition, etc., and can be determined by one skilled in the art.
[0137]
Cells into which the nucleic acid can be introduced for gene therapy purposes include any desired available cell type, including but not limited to: steroidogenic cells, adipocytes, epithelial cells, endothelium Cells, keratinocytes, fibroblasts, myocytes, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes; Stem or progenitor cells, especially hematopoietic stem or progenitor cells, such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver and the like. In a preferred embodiment, the cells used for gene therapy are adipocytes or steroidogenic cells. In another preferred embodiment, the cells used for gene therapy are autologous to the patient.
[0138]
In one embodiment where a recombinant cell is used for gene therapy, the nucleic acid sequence encoding perilipin is introduced into the cell so that it can be expressed by the cell or its progeny, and then the recombinant cell is treated with a therapeutic effect. Administer in vivo as indicated. In certain embodiments, stem or progenitor cells are used. Any stem and / or progenitor cell that can be isolated and maintained in vitro may be used in accordance with this embodiment of the invention (see, eg, PCT Publication WO 94/08598 (1994). April 28); Temple and Anderson, 1992, Cell 71: 973-985; Rheinwald, 1980, Meth. Cell Bio. 21A: 229; and Pittekow and Scott, 1986, MayoCl., 1986, MayoCl.
[0139]
In certain embodiments, the nucleic acid introduced for gene therapy purposes comprises a constitutive promoter operably linked to the coding region of perilipin, such that expression of the nucleic acid is constitutive. In another embodiment, the nucleic acid introduced for gene therapy purposes comprises an inducible promoter operably linked to the coding region of perilipin to control the presence or absence of a suitable transcription inducer. Allows the expression of the nucleic acid to be controlled. Using the examples of constitutive or inducible promoters described herein or known to those skilled in the art, regulate the expression of a nucleic acid molecule encoding perilipin or a fragment thereof or a perilipin fusion protein for gene therapy purposes. be able to.
[0140]
5.4. Perilipin antagonists or agonists
Described below are compounds that can function as either antagonists or agonists of one or more of the perilipin isoforms, depending on the particular application for which they are utilized.
[0141]
In a specific embodiment, the compound that functions as an antagonist or agonist of perilipin specifically binds to perilipin A, perilipin B, perilipin C, or any combination thereof (ie, as determined by immunoassays well known to those of skill in the art). And binds to related antigens with little or no cross-reactivity) and / or recognizes them. In another embodiment, the compound that functions as an antagonist or agonist of perilipin is one that specifically modulates the expression and / or activity of perilipin A, perilipin B, perilipin C, or any combination thereof.
[0142]
5.4.1. antibody
Utilizing antibodies that function as antagonists or agonists, disorders related to weight (eg, obesity), disorders of lipid metabolism (eg, lipodystrophy), disorders characterized by lipid accumulation (eg, atherosclerosis) or diabetes Can prevent, treat or ameliorate one or more of the signs or symptoms associated with Depending on the particular antibody, that antibody can function as an antagonist or agonist.
[0143]
An antibody that functions as an antagonist of one or more perilipin isoforms is an antibody that specifically binds to one or more perilipin isoforms and interferes with its action. For example, one such antibody may specifically bind to a perilipin isoform in a manner that does not activate the perilipin isoform but destroys its ability to bind to a natural ligand. It is possible. Such antibodies include, but are not limited to, polyclonal, monoclonal, humanized, human, Fab fragments, single chain antibodies, chimeric antibodies, and the like.
[0144]
An antibody that functions as an agonist of one or more perilipin isoforms can specifically bind to perilipin and, by binding, activate one or more perilipin isoforms directly or indirectly. A working antibody. For example, one such antibody is a form in which the perilipin isoforms function as if one of the endogenous ligands were bound thereby thereby activating one of the signaling pathways. Can bind to the perilipin isoform. Such antibodies include, but are not limited to, polyclonal, monoclonal, humanized, human, Fab fragments, single chain antibodies, chimeric antibodies, and the like.
[0145]
In one embodiment, the antibody used as an antagonist or agonist of perilipin specifically recognizes and / or binds perilipin A, perilipin B, perilipin C, or any combination thereof. If antibody fragments are used, the smallest inhibitory fragment that binds to perilipin is preferred. For example, a peptide having an amino acid sequence corresponding to the domain of the variable region of an antibody that binds to perilipin can be used. Such peptides can be synthesized chemically, or produced by recombinant DNA techniques, using methods well known in the art (see, eg, Creighton, 1983, supra; and Sambrook et al., 1989, supra). . Alternatively, a single-chain antibody that binds to an intracellular epitope, such as a neutralizing antibody, can be administered. Such single chain antibodies can be obtained, for example, by utilizing the techniques described by Marasco et al. (Marasco, W. et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7898-7893). Administration can be by expressing the nucleotide sequence encoding the antibody in a target cell population.
[0146]
In some embodiments, any commercially available antibody that specifically binds one or more perilipin isoforms or fragments thereof can be used in accordance with the present invention. One example of an antibody that specifically binds to perilipin is described in Research Diagnostics, Inc. (Flanders, NJ). Polyclonal guinea pig anti-perilipin antibody. In some other embodiments, commercially available antibodies that specifically bind to one or more perilipin isoforms or fragments thereof are not used in the present invention.
[0147]
5.4.1.1. Production of antibodies
One or more perilipin isoforms or fragments thereof can be used as an immunogen to generate antibodies that immunospecifically bind to one or more perilipin isoforms. Such immunogens can be isolated by any convenient means known in the art. Antibodies of the present invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F (ab ') fragments, Fab expression libraries. Included are fragments produced, anti-idiotype (anti-Id) antibodies, as well as any of the epitope binding fragments described above. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds one of the antigens. The immunoglobulin molecule can be of any class (eg, IgG, IgE, IgM, IgD and IgA) or subclass of the immunoglobulin molecule.
[0148]
Anti-perilipin antibodies include analogs and derivatives that have been modified (ie, any type of molecule is covalently attached), provided that such covalent attachment does not impair immunospecific binding. I do. For example, but not limited to, derivatives and analogs of antibodies include glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, cellular ligands Alternatively, those modified further by linking to other proteins are included. Any of a number of chemical modifications can be performed by known techniques. Includes, but is not limited to, specific chemical cleavage, acetylation, formylation, and the like. Further, the analog or derivative may include one or more non-classical amino acids.
[0149]
In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, for example, ELISA (enzyme linked immunosorbent assay). For example, to select antibodies that recognize a particular region of perilipin, the resulting hybridomas may be assayed for products that bind to perilipin fragments containing such domains. For example, in order to select an antibody that specifically binds to perilipin A but does not specifically bind to perilipin B or C (or weakly binds), positive binding to perilipin A and binding to perilipin B or C are considered. Can be selected based on the lack of (or low) binding of Thus, the present invention provides a higher affinity (preferably at least 2 fold, more preferably at least 5 fold, even more preferably at least 10 fold) affinity for a particular perilipin isoform than for another isoform of perilipin. To provide an antibody (preferably a monoclonal antibody).
[0150]
Polyclonal antibodies that can be used in the methods of the invention are a heterogeneous population of antibody molecules derived from the serum of the immunized animal. Unfractionated immune sera can also be used. Various techniques known in the art can be used for the production of polyclonal antibodies against perilipin isoforms or fragments thereof. In certain embodiments, a rabbit polyclonal antibody against an epitope of the perilipin isoform can be obtained. For example, for the production of polyclonal or monoclonal antibodies, various host animals, including but not limited to rabbits, mice, and the like, may be injected by injection of a native perilipin isoform or a fragment thereof or a synthetic (eg, recombinant) variant thereof. Rats and the like can be immunized. Depending on the species of the host, various adjuvants can be used to enhance the immunological response. These include, but are not limited to, complete or incomplete Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyholes Limpet hemocyanin, dinitrophenol, and adjuvants such as BCG (bacille Calmette-Guerin) or Corynebacterium parvum. Other adjuvants are well known in the art.
[0151]
For preparation of monoclonal antibodies (mAbs) specific for perilipin isoforms or fragments thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique first developed by Kohler and Milstein (1975, Nature 256: 495-497), and the trioma technique, the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and human monoclonal antibodies. There is the EBV hybridoma technique for production (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. License, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class and subclass, including IgG, IgM, IgE, IgA, IgD. The hybridoma producing the mAb of the present invention can be cultured in vitro or in vivo. In another embodiment of the present invention, monoclonal antibodies can be produced in sterile animals using known techniques (PCT / US90 / 02545, incorporated herein by reference).
[0152]
Monoclonal antibodies include, but are not limited to, human monoclonal antibodies and chimeric monoclonal antibodies (eg, human-mouse chimeras). A chimeric antibody is a molecule in which different portions are derived from different animal species, including those having a human immunoglobulin constant region and a variable region derived from a mouse mAb (eg, Cabilly et al., US Pat. 816,567; and Boss et al., US Pat. No. 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule from a non-human species that has one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule (see, for example, Queen, US Patent No. No. 5,585,089, which is incorporated herein by reference in its entirety).
[0153]
Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using the methods described below: PCT Publication WO 87/02671; European Patent Application 184,187: European Patent Application 171 496; European Patent Application 173 494; PCT Publication WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al., 1988, Science 240: 1041-1040; Liu et al., 1987, Proc. Natl. Acad. Sci. ScL USA 84: 3439-3443; Liu et al., 1987, J. Am. Immunol. 139: 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al., 1987, Canc. Res. 47: 999-1005; Wood et al., 1985, Nature 314: 446-449; Shaw et al., 1988, J. Am. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al., 1986, Bio / Techniques 4: 214; U.S. Patent No. 5,225,539; Jones et al., 1986, Nature 321: 552-525. Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al., 1988, J .; Immunol. 141: 4053-4060.
[0154]
Antibodies that are fully human are particularly desirable for therapeutic treatment of human subjects. Such antibodies can be produced using transgenic mice. Such mice are not capable of expressing endogenous immunoglobulin heavy and light chain genes, but are capable of expressing human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, eg, all or part of a perilipin isoform. Monoclonal antibodies specific for this antigen can be obtained using conventional hybridoma technology. The transgenes of human immunoglobulins carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technique for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). For a detailed discussion of this technique for the production of human and human monoclonal antibodies and the protocols for producing these antibodies, see, for example, US Pat. No. 5,625,126; US Pat. No. 5,633. U.S. Patent No. 5,569,825; U.S. Patent No. 5,661,016; and U.S. Patent No. 5,545,806. Further, Abgenix, Inc. Companies such as (Freemont, CA) and Genpharm (San Jose, CA) can order and supply specific human antibodies to a given antigen using techniques similar to those described above.
[0155]
Fully human antibodies that recognize a given epitope can be made using a technique referred to as "guided selection". In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, can be used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al. (1994) Bio / technology 12: 899-). 903).
[0156]
Anti-perilipin antibodies can also be made using various phage display methods known in the art. In the phage display method, a functional antibody domain is displayed on the surface of a phage particle carrying a polynucleotide sequence encoding the domain. In particular, such phage can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. Phage used in these methods are typically fd and M13 expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. Filamentous phage containing a binding domain. Phage display methods that can be used to produce the antibodies of the present invention include those disclosed below: Brinkman et al. Immunol. Methods 182: 41-50 (1995); Ames et al. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application number PCT / GB91 / 01134; PCT publication WO 90 /. WO809 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733, 43; and 5,969,108; incorporated herein by reference each of them as a whole.
[0157]
Following phage selection, the antibody coding region from the phage is isolated and used to generate whole antibodies, including human antibodies, or other desired antigen-binding fragments, as described in the above references. For example, as described in detail below, it can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria. For example, Fab, Fab 'and F (ab')₂Techniques for recombinantly producing fragments can also use methods known in the art, such as those disclosed below: PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12 (6): 864. -869 (1992); Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988), the entirety of which are incorporated herein by reference.
[0158]
Examples of techniques that can be used to produce single-chain Fvs and antibodies include those described below: US Pat. Nos. 4,946,778 and 5,258,498. Huston et al., Methods in Enzymology 203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988).
[0159]
The present invention further provides for the use of bispecific antibodies. It can be manufactured by methods known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, the two chains having different specificities (Milstein et al., 1983, Nature 305: 537-539). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) potentially produce a mixture of ten antibody molecules, of which only one is the correct bispecific Sexual structure. Purification of the correct molecule (usually performed by an affinity chromatography step) is rather cumbersome and results in low product yields. Similar procedures are described in WO 93/08829 (published May 13, 1993) and Traunecker et al., 1991, EMBO J. 10: 3655-3659.
[0160]
According to another more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably has an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferable that the first heavy chain constant region (CH1) containing a site necessary for light chain binding be present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This method is a highly versatile way to adjust the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide the optimal yield. However, if high yields are obtained as a result of the expression of at least two polypeptide chains in equal proportions, or where the proportion is not particularly important, two or all of the coding sequences of the three polypeptide chains Can be inserted into one expression vector.
[0161]
In a preferred embodiment of this technique, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (primary) in the other arm. 2 providing binding specificity). This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. Because the immunoglobulin light chain is present in only half of the bispecific molecule, an easy separation method is possible. This approach is disclosed in WO 94/04690 (published March 3, 1994). For further details for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121: 210.
[0162]
The present invention provides functionally active fragments, derivatives or analogs of anti-perilipin immunoglobulin molecules. “Functionally active” refers to an anti-anti-idiotype antibody (ie, tertiary) in which a fragment, derivative or analog recognizes the same antigen as is recognized by the antibody from which the fragment, derivative or analog is derived. Antibody). Specifically, in a preferred embodiment, the idiotypic antigenicity of the immunoglobulin molecule is enhanced by deleting the framework and CDR sequences C-terminal to the CDR sequence that specifically recognizes the antigen. . To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in any of the binding assays known in the art.
[0163]
The present invention relates to F (ab ')₂Antibody fragments such as fragments and Fab fragments are provided. Antibody fragments that recognize specific epitopes can be prepared by known techniques. F (ab ')₂Fragments are composed of the variable region, the light chain constant region and the CH1 domain of the heavy chain, and can be made by pepsin digestion of the antibody molecule. Fab fragment is F (ab ')₂Prepared by reducing the disulfide bridges of the fragment. The present invention relates to heavy and light chain dimers of the antibodies of the present invention, or any minimal fragment thereof, such as Fv or single chain antibodies (SCA) (eg, as described below: US Pat. 778; Bird, 1988 Science 242: 423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-54) or the present invention. Any other molecule having the same specificity as the antibody of the present invention is also provided. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region by an amino acid bridge into a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can be used (Skerra et al., 1988, Science 242: 1038-1041).
[0164]
In another embodiment, the invention provides a fusion protein of an anti-perilipin antibody (or a functionally active fragment thereof). For example, in this case, the antibody is covalently linked (eg, at the N-terminus or C-terminus) to the amino acid sequence of another protein that is not an antibody (or a portion thereof, preferably at least 10, 20, or 50 amino acid portions of the protein) (Peptide bonds). Preferably, the immunoglobulin or fragment thereof is covalently linked to another protein at the N-terminus of the constant domain. Such a fusion protein can facilitate purification or increase in vivo half-life.
[0165]
5.4.1.2. Antibody expression
Anti-perilipin antibodies can be produced by any method known in the art for antibody synthesis, particularly by chemical synthesis or recombinant expression, and are preferably produced by recombinant expression techniques.
[0166]
Recombinant expression of an antibody or a fragment, derivative or analog thereof requires the construction of a nucleic acid encoding the antibody. If the nucleotide sequence of the antibody is known, the nucleic acid encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al., 1994, BioTechniques 17: 242). Briefly, this involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence by PCR, annealing and ligation of these oligonucleotides, and subsequent amplification of the ligated oligonucleotides.
[0167]
Alternatively, a nucleic acid encoding an antibody can be obtained by cloning the antibody. Even though clones containing the nucleic acid encoding the particular antibody are not available, if the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be obtained from a suitable source (eg, an antibody cDNA library, or a cDNA library thereof). CDNA libraries made from either antibody expressing tissues or cells), by PCR amplification using synthetic primers capable of hybridizing to the 3 'and 5' ends of the sequence or specific to that particular gene sequence Can be obtained by cloning using a specific oligonucleotide probe.
[0168]
If antibody molecules that specifically recognize a particular perilipin isoform (or a source of a cDNA library for cloning nucleic acid encoding such an antibody) are not available, a specific perilipin isoform specific Antibodies can be produced by any method known in the art, for example, by immunizing an animal such as a rabbit to produce a polyclonal antibody, or more preferably by producing a monoclonal antibody. Alternatively, one clone encoding at least the Fab portion of the antibody is replaced with a clone of a Fab fragment that binds a Fab expression library to a specific antigen (eg, as described in Huse et al., 1989, Science 246: 1275-1281). Or screening antibody libraries (see, eg, Clackson et al., 1991, Nature 352: 624; and Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937). ).
[0169]
After obtaining a nucleic acid encoding at least the variable domain of the antibody molecule, it can be introduced into a vector containing a nucleotide sequence encoding the constant region of the antibody molecule (eg, PCT Publication No. WO 86/05807). PCT Publication No. WO 89/01036; and US Pat. No. 5,122,464). Vectors containing complete light or heavy chains that are capable of co-expressing the nucleic acid to express a complete antibody molecule are also available. The nucleic acid encoding this antibody is then used to replace (or delete) one or more cysteine residues in the variable region involved in intrachain disulfide bonds with amino acid residues that do not contain a sulfhydryl group. Required nucleotide substitutions or deletions can be introduced. Such modifications can be made by any method known in the art for introducing a particular mutation or deletion within a single nucleotide sequence. Examples include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551), PCT-based methods, and others.
[0170]
Moreover, techniques developed for the production of "chimeric antibodies" by splicing the gene of a mouse antibody molecule with appropriate antigen specificity together with the gene of a human antibody molecule with appropriate biological activity ( Morrison et al., 1984, Proc. Natl. Acad. Sci. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454) can be used. As described in the above literature, a chimeric antibody is a molecule in which different portions are derived from different animal species and has a variable region derived from a mouse mAb and a human antibody constant region, such as a humanized antibody. is there.
[0171]
Once a nucleic acid encoding an anti-perilipin antibody molecule has been obtained, a vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein of the invention by expressing a nucleic acid containing an antibody molecule sequence are described herein. Methods that are well known to those of skill in the art can be used to construct expression vectors containing antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. For example, Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA), and Ausubel et al. See the described technique.
[0172]
The expression vector is introduced into host cells by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
[0173]
The host cells used to express the recombinant antibodies of the present invention may be any of bacterial cells, Escherichia coli, or the like, or preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecules. Good. In particular, the combination of a mammalian cell, such as Chinese hamster ovary cell (CHO), and a vector, such as a major intermediate early gene promoter element from human cytomegalovirus, is an effective expression system for antibodies ( Foecking et al., 198, Gene 45: 101; Cockett et al., 1990, Bio / Technology 8: 2).
[0174]
A variety of host-expression vector systems are available for expressing anti-perilipin antibody molecules. Such a host-expression system not only refers to a vehicle that can produce and purify the coding sequence of interest, but also to express the antibody molecule of the present invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. Also refers to cells that can These include, but are not limited to: bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing a sequence encoding the antibody, such as E. coli (E. microorganisms such as E. coli and B. subtilis; yeast transformed with a recombinant yeast expression vector containing a sequence encoding the antibody (eg, Saccharomyces, Pichia); and encoding the antibody An insect cell line infected with a recombinant virus expression vector containing the sequence (eg baculovirus); with a recombinant virus expression vector containing the sequence encoding the antibody (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) Infected Or a plant cell line transformed with a recombinant plasmid expression vector (eg, a Ti plasmid); or a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) or a promoter derived from a mammalian virus (eg, an adenovirus late A mammalian cell line (eg, COS, CHO, BHK, 293, 3T3 cells) carrying a recombinant expression construct containing the promoter, vaccinia virus 7.5K promoter.
[0175]
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, if one wishes to produce such proteins in large quantities for the production of a pharmaceutical composition containing the antibody molecule, a vector that directs high-level expression of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the following: E. coli expression vector pUR278 (Rother et al., 1983, EMBO J. 2: 1791), in which case the sequence encoding the antibody is contained in the vector lacZ. The fusion proteins are produced by ligation individually in frame with the coding region; the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; and Van Heake & Schuster, 1989, J. Biol. Chem. 24: 5503-5509); The pGEX vector can also be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix followed by elution in the presence of free glutathione. The pGEX vector can be designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product is released from the GST moiety.
[0176]
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing a foreign gene. This virus grows in Spodoptera frugiperda cells. Sequences encoding the antibody can be cloned individually into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems (eg, adenovirus expression systems) can be utilized.
[0177]
As discussed above, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product, in the specific fashion desired. Modification (eg, glycosylation) and processing (eg, cleavage) of such protein products can be important for the function of the protein.
[0178]
For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, a cell line that stably expresses the antibody of interest can be prepared by transfecting cells with an expression vector containing the nucleotide sequence of the antibody and the nucleotide sequence of a selectable marker (eg, neomycin or hygromycin), and expressing the selectable marker. By selecting, it can be produced. Such engineered cell lines may be particularly useful in screening and evaluating compounds that interact directly or indirectly with the antibody molecule.
[0179]
Expression levels of antibody molecules can be increased by amplification of the vector (for a review, see Bebbington and Hentschel, "Use of a vector based on gene amplification for expression of cloned genes in mammalian cells in DNA cloning ( The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning. ", Vol. 3, Academic Press, Academic Pressing, 87. If the marker in the antibody expressing vector system is amplifiable, increasing the level of inhibitor present in the culture of host cells will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).
[0180]
A host cell may be co-transfected with two expression vectors of the present invention, wherein the first vector encodes a polypeptide derived from the heavy chain and the second vector encodes a polypeptide derived from the light chain. . Including the same selectable marker in the two vectors allows for equivalent expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both the heavy and light chain polypeptides may be used. In such situations, the light chain must be placed in front of the heavy chain to avoid excess toxic free heavy chains (Proudfoot, 1986, Nature 322: 52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77: 2197). The coding sequences for the heavy and light chains may consist of cDNA or genomic DNA.
[0181]
After recombinantly expressing the antibody molecule of the present invention, the antibody molecule can be purified by any method known in the art for purification of an antibody molecule. For example, chromatography (e.g., ion exchange chromatography, affinity chromatography with protein A or specific antigens, and size column chromatography), centrifugation, differential solubility, or any other for protein purification. According to standard techniques.
[0182]
Alternatively, any fusion protein can be easily purified by utilizing an antibody specific to the expressed fusion protein. For example, the system described by Janknecht et al. Allows for the immediate purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-897). ). In this system, the gene of interest is subcloned into a vaccinia recombinant plasmid such that the open reading frame of this gene is translated by fusing it to an amino-terminal tag consisting of six histidine residues. This tag acts as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus were Ni²⁺Load onto a nitriloacetic acid-agarose column and selectively elute histidine-tagged proteins with imidazole containing buffer.
[0183]
5.4.1.3. Conjugate antibody
In one preferred embodiment, the anti-perilipin antibody or fragment thereof is conjugated to a diagnostic or therapeutic moiety. Antibodies can be used for diagnosis or for determining the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody and a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for positron emission tomography), and non-radioactive paramagnetic metal ions It is. See US Pat. No. 4,741,900 for a general discussion of metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Preferred enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase, preferred prosthetic groups include streptavidin, avidin and biotin, and preferred fluorescent materials include Umbellium Ferron, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin, suitable luminescent materials include luminol, and suitable bioluminescent materials include luciferase, luciferin and aequorin. Included and suitable radionuclides include:¹²⁵I,¹³¹I,¹¹¹In and⁹⁹Tc is included.
[0184]
Anti-perilipin antibodies or fragments thereof can be conjugated to a therapeutic agent or drug moiety to modify a given biological response. It is not intended that the therapeutic agent or drug moiety be limited to traditional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that possesses the desired biological activity. Such proteins can include, for example: toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; tumor necrosis factor, interferon-α, interferon-β, nerve growth factor, platelet-derived growth factor Tissue plasminogen activator, thrombotic or anti-angiogenic agents, such as angiostatin or endostatin; or lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin Modification of biological response such as leukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors Agent.
[0185]
Techniques for conjugating such therapeutic moieties to antibodies are well known, for example, see Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer. Therapy) ", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al. (Eds.). 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: Online Review of Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review of Pharmacy." Applications, Pinchera et al. (Eds.), Pp. 475-506 (1985); "Analysis, Results, and Future Prospects of Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy (Analysis, Results, And Future Prospective of The Therapeutic Use of Radioactive Antibiotics, Inc.). For Cancer Detection And Therapy, Baldwin et al. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev .. , 62: 119-58 (1982).
[0186]
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate, as described by Segal in US Pat. No. 4,676,980.
[0187]
Antibodies conjugated or unconjugated to a therapeutic moiety can be used alone or as a therapeutic, administered in combination with cytotoxic factors and / or cytokines.
[0188]
5.5. Methods of treating disorders associated with inappropriate regulation of body weight and / or lipid metabolism
The present invention relates to the treatment of weight disorders such as obesity, inappropriate lipid metabolism by the administration of one or more pharmaceutical compositions comprising one or more compounds that modulate the expression and / or activity of one or more perilipin isoforms. Provide prevention, treatment or amelioration of a disorder characterized by (eg, lipodystrophy), a disorder characterized by lipid accumulation (eg, atherosclerosis), and one or more signs or symptoms associated with diabetes. In one preferred embodiment, a pharmaceutical composition is administered to a subject (ie, an animal) for the prevention, treatment or amelioration of one or more signs or symptoms associated with obesity. In another preferred embodiment, a pharmaceutical composition is administered to a subject (ie, an animal) for the prevention, treatment or amelioration of one or more signs or symptoms associated with diabetes (eg, diabetes mellitus type 1 or 2). For these embodiments, the pharmaceutical compositions comprise one or more compounds that antagonize the activity and / or expression of one or more perilipin isoforms, and a pharmaceutically acceptable carrier.
[0189]
In another preferred embodiment, the pharmaceutical composition is used to prevent, treat or ameliorate one or more signs or symptoms associated with a disorder characterized by weight loss, such as cachexia, bulimia and anorexia. That is, it is administered to an animal, preferably a mammal, and more preferably a human. For this embodiment, the pharmaceutical composition comprises one or more compounds that agonize the activity and / or expression of one or more perilipin isoforms, and a pharmaceutically acceptable carrier.
[0190]
In certain embodiments, the subject is administered the following amount of a composition of the present invention: an amount effective to increase lipid metabolism; or an amount effective to increase muscle mass; or an amount to increase body weight; Or an amount that reduces body fat; or an amount effective to increase insulin secretion; or an amount effective to reduce insulin resistance; or an amount effective to reduce glucose intolerance; or obesity, inappropriate An amount effective to prevent, treat or ameliorate one or more signs or symptoms associated with a disorder characterized by severe lipid metabolism, a disorder characterized by lipid accumulation, or diabetes. In certain other embodiments, the subject is administered the following amount of a composition of the present invention: treatment of one or more signs or symptoms associated with a disorder characterized by weight loss, such as cachexia and anorexia; An amount effective to prevent or ameliorate; or an amount effective to increase body fat; or an amount effective to increase body weight; or an amount effective to increase lipid accumulation.
[0191]
The present invention provides methods for the treatment, prevention or amelioration of one or more signs or symptoms associated with weight disorders and / or inappropriate lipid metabolism, which involve the effects of fat localization. Thus, the methods of the present invention have a reduced risk of adverse side effects observed for other targets for the treatment of weight disorders such as obesity.
[0192]
In certain embodiments, used to prevent, treat or ameliorate one or more signs or symptoms associated with weight disorders (eg, obesity), disorders characterized by inappropriate lipid metabolism, disorders characterized by lipid accumulation, and diabetes. The compounds identified according to the methods of the present invention are those that are not known or have not been previously used in the prevention, treatment or amelioration of such disorders. In another embodiment, used in the prevention, treatment or amelioration of one or more signs or symptoms associated with weight disorders (eg, obesity), disorders characterized by inappropriate lipid metabolism, disorders characterized by lipid accumulation, and diabetes. The compound identified according to the method of the invention preferentially affects the expression or activity of one perilipin isoform and does not affect the expression or activity of the other two isoforms. is there. In another embodiment, used in the prevention, treatment or amelioration of one or more signs or symptoms associated with weight disorders (eg, obesity), disorders characterized by inappropriate lipid metabolism, disorders characterized by lipid accumulation, and diabetes. The compounds identified according to the methods of the invention are those that preferentially affect perilipin A expression or activity.
[0193]
5.6. How to enhance livestock and poultry
The present invention relates to a method for enhancing the weight and / or behavior of an animal, preferably on companion animals (eg dogs, cats and horses), livestock (eg cows, horses and pigs) and poultry (eg chickens and turkeys). And a composition. The invention also relates to the use of preferably companion animals (eg dogs, cats and horses), livestock (eg cows, horses and pigs) and poultry (eg chickens and turkeys) for the prevention or treatment of weight disorders such as obesity in animals. Also provided are methods and compositions for The present invention also relates to a companion animal (preferably a companion animal) for the prevention, treatment or amelioration of one or more signs or symptoms associated with disorders characterized by inappropriate lipid metabolism, disorders characterized by lipid accumulation or diabetes in animals. Methods and compositions are provided for, for example, dogs, cats and horses), livestock (eg, cows, horses and pigs), and poultry (eg, chickens and turkeys). The present invention further relates to methods and compositions for improving animal health, preferably on companion animals (eg dogs, cats and horses), livestock (eg cows, horses and pigs) and poultry (eg chickens and turkeys). .
[0194]
In one embodiment, livestock or poultry are administered a pharmaceutical composition comprising one or more antagonists of one or more perilipin isoforms to enhance their body weight and / or behavior. In another embodiment, livestock or poultry are administered a pharmaceutical composition comprising one or more antagonists of one or more perilipin isoforms to enhance their lipid metabolism and / or increase muscle mass. In another embodiment, one or more perilipin isoforms are used in livestock or poultry to treat, prevent or ameliorate one or more signs or symptoms associated with obesity, disorders of lipid metabolism, disorders characterized by lipid accumulation, or diabetes. A pharmaceutical composition comprising one or more antagonists of the foam is administered. In yet another embodiment, a livestock or poultry is treated with one or more perilis for the treatment, prevention or amelioration of one or more signs or symptoms associated with a weight loss characterized by weight loss such as cachexia or anorexia. A pharmaceutical composition comprising one or more agonists of the pin isoform is administered.
[0195]
5.7. Pharmaceutical formulations and routes of administration
The present invention provides a therapeutic (and prophylactic) method comprising administering to a subject an effective amount of a compound of the present invention. In one preferred embodiment, the compound is purified substantially (eg, so as to be substantially free of substances that limit its effect or produce undesirable side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, most preferably a human. In one particular embodiment, the poultry or non-human mammal is the subject or animal. In another specific embodiment, the subject or animal is a human.
[0196]
While the formulations and methods of administration that can be utilized when the compound comprises a nucleic acid are described above, other suitable formulations and routes of administration are described below.
[0197]
A variety of delivery systems are known and can be used to administer the compounds of the invention. For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-). 4432), construction of nucleic acids as part of retroviruses or other vectors, and others. Methods of introduction can be enteral and parenteral and include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral routes. The compounds may be administered by any convenient route, eg, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (eg, oral, rectal and intestinal mucosa, etc.) and along with other biologically active agents. May be administered. Administration can be systemic or local.
[0198]
In one particular embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; This can be achieved, for example, but not limited to, by local injection during surgery, by local application, such as by injection, by catheter, or by implant. The implant may be a porous, non-porous, or gelatinous material, including a membrane, such as a siaastic membrane, or a fiber.
[0199]
In another embodiment, the compounds can be delivered in vesicles, particularly in liposomes (eg, Langer, 1990, Science 249: 1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer-, Canada). See Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berstein, ibid., Pp. 317-327;
[0200]
In yet another embodiment, the compounds can be delivered in a sustained or controlled release system. In one embodiment, a pump can be used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgary 88: 507; Saudek et al., 1989, N. Engl.J.Med.321: 574). In another embodiment, a polymeric material can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida, Canada, 1974; Controlled Drug PharmaBridge, Canada). Smolen and Ball (eds), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228: 190; 1989, Ann. ol.25: 351; and Howard et al., 1989, J.Neurosurg.71: See 105). In yet another embodiment, a sustained or controlled release system can be placed near the therapeutic target, ie, in adipose tissue, thereby requiring only a small portion of the systemic dose (eg, Goodson, Medical Applications of Controlled). Release, supra, Vol. 2, pp. 115-138 (1984)).
[0201]
Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533).
[0202]
In one particular embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid is constructed as part of a suitable nucleic acid expression vector, such that it becomes intracellular, for example as follows. Can be administered in vivo to enhance expression of the protein encoded by this nucleic acid: use of retroviral vectors (see US Pat. No. 4,980,286); direct injection; Use of bombardment of microparticles (eg, a gene gun; Biolistic, Dupont) or coating with lipids or cell surface receptors or transfectants; administration linked to a homeobox-like peptide known to enter the nucleus (eg, Joliot et al., 1991, Proc. Natl. Aca. .Sci.USA 88: 1864-1868 reference), and others. Alternatively, a nucleic acid can be introduced into a cell and incorporated into host cell DNA for expression by homologous recombination.
[0203]
The present invention also provides a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In one particular embodiment, the term “pharmaceutically acceptable” has been approved by federal or state government regulators for use in animals, and more particularly in humans, or has been approved by the United States Pharmacopoeia or other generally It means that it is listed in a recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, glycol, water, ethanol and the like are included. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in W. Martin's "Remington's Pharmaceutical Sciences". The inclusion of a therapeutically effective amount of the compound in such a composition, preferably in purified form, together with a suitable amount of carrier will provide the subject with a form suitable for administration. The formulation must suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions of the present invention are sterile.
[0204]
In one preferred embodiment, the compositions are formulated according to conventional procedures, as pharmaceutical compositions adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the components are mixed separately or as a lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachette indicating the amount of active agent, or mixed in unit dosage form. Supplied. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0205]
The compounds of the present invention can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with free carboxyl groups, such as sodium and potassium. , Ammonium, calcium, iron (III) hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, those derived from procaine and the like.
[0206]
The toxicity and therapeutic efficacy of the compound can be measured, for example, by LD₅₀(50% lethal dose in population) and ED₅₀Determination of (50% therapeutically effective amount of the population) can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index and the ratio LD₅₀/ ED₅₀Can be expressed as Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but design delivery systems that target such compounds to diseased tissue sites to minimize the risk of damaging uninfected cells and thereby reduce side effects So, care must be taken.
[0207]
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans and the frequency of dosage of a given dosage. The dosage of such compounds is preferably such that the ED has little or no toxicity.₅₀Within the circulating concentration range including Dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. IC determined in cell culture₅₀Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the (ie, the concentration of the candidate compound that achieves half-maximal suppression of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.
[0208]
Suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kg of body weight. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight. Oral formulations preferably contain 10% to 95% of the active ingredient.
[0209]
For antibodies, the preferred dosage is 0.1 mg / kg to 125 mg / kg (more preferably 0.1 mg / kg to 75 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 0.1 mg / kg). 1 mg / kg to 25 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 15 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / Kg-5 mg / kg, 0.1 mg / kg-2.5 mg / kg, or 0.1 mg / kg-1 mg / kg). Generally, partially human antibodies and fully human antibodies have a longer half-life in the human body than other antibodies. Thus, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize the antibody and to enhance uptake and tissue penetration. Methods for lipidation of antibodies are described in Cruikshank et al., 1997, J. Am. Acquired Immunity Definity Syndromes and Human Retrovirology 14: 193.
[0210]
A therapeutically effective amount of a protein or polypeptide (ie, an effective dose or effective dose) is about 0.001-30 mg / kg body weight, preferably about 0.01-25 mg / kg body weight, more preferably Is in the range of about 0.1-20 mg / kg body weight, and more preferably about 1-10 mg / kg body weight, 2-9 mg / kg body weight, 3-8 mg / kg body weight, 4-7 mg / kg body weight. kg body weight, or in the range of 5-6 mg / kg body weight.
[0211]
One skilled in the art will appreciate that certain factors can affect the dosage required to effectively treat a subject. These include, but are not limited to, the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other existing diseases. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is dosed once a week with an antibody, protein or polypeptide in the range of about 0.1-20 mg / kg body weight for about 1-10 weeks, preferably 2-8 weeks, more preferably Treat for about 3 to 7 weeks, and even more preferably for about 4, 5 or 6 weeks. It will also be appreciated that the effective dosage of the antibody, protein or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Variations in dosage can also be apparent to one skilled in the art from the results of the diagnostic assays described herein.
[0212]
It is understood that the appropriate dose of a small molecule drug will depend on a number of factors which are within the knowledge of the ordinarily skilled practitioner, veterinarian or researcher. The dose of the small molecule will depend, for example, on its identity, size and the condition of the subject or sample to be treated, and, if applicable, the route of administration of the composition, and the nucleic acid or nucleic acid of the present invention which the practitioner seeks for the small molecule. It will vary depending on the effect on the polypeptide. Typical doses include milligrams or micrograms of small molecule per kg of subject or sample weight (eg, about 1 μg / kg to about 500 mg / kg, about 1 μg / kg to about 250 mg / kg, about 1 μg / Kg to about 100 mg / kg, about 1 μg / kg to about 50 mg / kg, about 1 μg / kg to about 25 mg / kg, about 1 μg / kg to about 10 mg / kg, about 1 μg / kg to about 5 mg / kg. kg, about 1 μg / kg to about 1 μg / kg, about 1 μg / kg to about 500 μg / kg, about 1 μg / kg to about 250 μg / kg, about 1 μg / kg to about 100 μg / kg, about 1 μg / kg to about 50 μg / kg. kg, about 1 μg / kg to about 25 μg / kg, or about 1 μg / kg to about 10 μg / kg). It will be appreciated that the appropriate dose of a small molecule will depend on the potency of the small molecule for modulating expression or activity. Such appropriate doses are known in the art or can be determined using the assays described herein. When one or more small molecules are administered to an animal (eg, a human) to modulate the expression or activity of one or more perilipin isoforms, a practitioner, veterinarian, or researcher may, for example, initially use a relatively low dose. And then escalate the dose until an appropriate response is obtained. Furthermore, the dose level specified for any particular animal subject will depend on the activity of the particular compound used, the age, weight, general health, gender and diet of the subject, time of administration, route of administration, rate of elimination. It will be appreciated that combination with any agent, as well as the degree of expression or activity of one or more modulating perilipin isoforms, will be appreciated.
[0213]
The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Optionally, these containers may be accompanied by a notice in the form prescribed by a governmental agency that regulates the manufacture, use or sale of the pharmaceutical or biological product. This notice states (a) regulatory approval for manufacture, use or sale for administration to humans, (b) instructions for use, or both.
[0214]
5.8. Methods for assessing therapeutic utility
The present invention also aims to identify or demonstrate efficacy of compounds for the treatment or prevention of weight disorders (eg, obesity, cachexia and anorexia), lipid metabolism disorders, disorders characterized by lipid accumulation, and diabetes. Also provided are assays for use in drug discovery. Candidate compounds can be assayed for their ability to modulate perilipin expression levels in subjects with weight impairment to the levels found in subjects without such impairment. Compounds capable of restoring expression levels of one or more perilipin isoforms in subjects with weight disorders characterized by weight loss to levels found in subjects without weight disorders, It can be used as a lead compound or used in therapy. Further, compounds capable of reducing the level of expression of one or more perilipin isoforms in a subject with a weight disorder characterized by weight gain (eg, obesity) to levels found in subjects without this weight disorder, It can be used as a lead compound for further drug discovery or used in therapy. Expression of perilipin isoforms may be determined by immunoassay, visualization after gel electrophoresis, detection of perilipin phosphorylation, detection of perilipin activity, or any other method taught herein or known to one of skill in the art. Can be assayed. Such assays can be used to screen for candidate drugs for clinical monitoring or drug development if the abundance of perilipin isoforms can serve as a surrogate marker for clinical disorders.
[0215]
In various specific embodiments, performing in vitro assays on cells representative of the cell type involved in a disorder to determine whether a compound has a desired effect on such cell types. it can. For example, steroid-synthesizing cells and adipocytes from animals with weight disorders such as obesity can be used to determine whether a compound has a desired effect on such cells.
[0216]
Before testing compounds for use in therapy in humans, they can be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, rabbits and the like. For in vivo testing, any of the animal model systems known in the art can be used prior to administration to humans. Examples of animal models of weight impairment include, but are not limited to, the following obesity models: leptin-resistant animals (eg, db / db mice), melanocortin-4-receptor knockout mice (MR-4)^{− / −}), Leptin-deficient mouse (ob / ob), tuby mouse (tubby protein deficient), fa / fa (Zucker Diabetical Fatty or ZDF) rat, melanocortin-3-receptor knockout mouse, POMC-deficient mouse, and fat / fat mouse ( For example, Barsh et al., 2000, Nature 404: 644-651; Fisher et al., 1999, Int. J. Obes. Rel. Metab. Disord. 23 Suppl: 54-58; Giridharan, 1998, Indian J. Med. : 225-242; Zhang et al., 1994, Nature 372: 425-432; Noben-Trauth et al., 1996, Nature 380: 534-538; Iid. a et al., 1996, BBRC 224: 597-604; Phillips et al., 1996, Nature Genetics 13: 18-19; Chen et al., 2000, Nature Genetics 26: 97-102; Butler et al., 2000, Endocrinology 141; Yawen et al., 1999, Nature Medicine 5: 1066-1070; and Naggert et al., 1995, Nature Genetics 10: 135-142). It will also be apparent to one of skill in the art, based on the present disclosure, to produce transgenic mice having a “knockout” mutation in the gene encoding perilipin. A “knockout” mutation in one gene is a mutation in which the mutated gene is not expressed, or is expressed at an abnormal or low level, resulting in little or no activity associated with the gene product. . Preferably, the transgenic animal is a mammal, more preferably, the transgenic animal is a mouse.
[0217]
In one embodiment, a candidate compound that modulates the level or expression of a perilipin isoform is a human subject having a weight disorder such as obesity, cachexia or anorexia, a disorder of lipid metabolism, a disorder characterized by lipid accumulation, or diabetes. Identify or demonstrate. According to this embodiment, a test for perilipin expression is performed by administering a candidate or control compound to a human subject and analyzing the expression of perilipin or the mRNA encoding the same in a biological sample (eg, serum or plasma). Determine the effect of the compound. By comparing the level of one or more perilipin isoforms or mRNA encoding the same in a subject or group of subjects treated with a control compound to that of a subject or group of subjects treated with a candidate compound, Candidate compounds that alter the expression of the isoform can be identified. Alternatively, altering the expression of one or more perilipin isoforms by comparing the level of one or more perilipin isoforms or mRNA encoding the same in a subject or group of subjects before and after administration of the candidate compound can do. Biological samples can be obtained and analyzed for mRNA or protein expression using techniques known to those skilled in the art.
[0218]
In another embodiment, candidate compounds that modulate the activity of perilipin are identified or demonstrated in a human subject with a disorder of weight, disorder of lipid metabolism, a disorder characterized by lipid accumulation or diabetes. According to this embodiment, the candidate or control compound is administered to a human subject and the effect of the candidate compound on the activity of perilipin is determined. By comparing a biological sample from a subject treated with a control compound to a sample from a subject treated with the candidate compound, candidate compounds that alter the activity of one or more perilipin isoforms can be identified. Alternatively, alterations in the activity of one or more perilipin isoforms can be identified by comparing the activity of one or more perilipin isoforms in a subject or group of subjects before and after administration of a candidate compound. Perilipin activity can be assessed as follows: detection of perilipin phosphorylation; induction of perilipin cell signaling pathways in biological samples (eg, serum or plasma) (eg, intracellular Ca²⁺, Diacylglycerol, IP3, etc.); detection of the activity of an enzyme whose activity is regulated by perilipin (eg, hormone-sensitive lipase activity); detection of a reporter gene or cellular response, eg, induction of lipid metabolism (eg, triacylglycerol, By detecting changes in the levels of non-esterified fatty acids or β-hydroxybutyric acid). Techniques known to those skilled in the art can be used to detect altered phosphorylation of perilipin, altered induction of second messenger of perilipin, or altered cellular response. For example, RT-PCR can be used to detect changes in second messenger induction of cells, and Western blot analysis after immunoprecipitation to detect changes in perilipin phosphorylation. be able to.
[0219]
In a preferred embodiment, a candidate compound that alters the level of expression of one or more perilipin isoforms to a level detected in a control subject (eg, a human without weight loss) is used for subsequent testing or therapeutic use. Choose for In another preferred embodiment, candidate compounds that alter the activity of one or more perilipin isoforms toward the activity seen in a control subject (eg, a human without weight loss) are identified for subsequent testing or therapeutic use. To choose.
[0220]
In another embodiment, candidate compounds are identified that reduce the severity of one or more signs or symptoms associated with weight loss in a human subject with weight loss. In accordance with this embodiment, the candidate or control compound is administered to a human subject with a weight disorder and the effect of the candidate compound on one or more signs or symptoms of the weight disorder is determined. By comparing a subject treated with a control compound to a subject treated with a test compound, candidate compounds that reduce one or more signs or symptoms can be identified. To determine whether a candidate compound reduces one or more signs or symptoms associated with weight loss, techniques known to practitioners familiar with weight loss can be used. For example, for the treatment of obese subjects, candidate compounds that increase lipid metabolism would be beneficial.
[0221]
In another embodiment, in a human subject having diabetes, reduce blood glucose levels, increase insulin sensitivity, increase insulin secretion, reduce the required dose of other antidiabetic drugs, or reduce diabetes. Identify candidate compounds that reduce the severity of one or more signs or symptoms involved. In accordance with this embodiment, a candidate or control compound is administered to a human subject with diabetes, and blood glucose, insulin sensitivity, insulin secretion, required doses of other antidiabetic agents, or candidates for one or more signs or symptoms of diabetes. Determine the effect of the compound. Reduce blood glucose levels, increase insulin sensitivity, reduce required doses of other antidiabetic drugs, or relate to diabetes by comparing a subject treated with a control compound with a subject treated with a test compound. Candidate compounds that reduce one or more signs or symptoms of the disease can be identified. To determine whether a candidate compound reduces one or more signs or symptoms associated with diabetes, techniques known to a practitioner familiar with diabetes can be used.
[0222]
In one preferred embodiment, candidate compounds that reduce the severity of one or more signs or symptoms associated with weight loss in a human with weight loss are selected for subsequent testing or therapeutic use. In another preferred embodiment, candidate compounds that reduce the severity of one or more signs or symptoms associated with diabetes in a human with diabetes are selected for subsequent testing or therapeutic use.
[0223]
5.9. Diagnostic and monitoring techniques
According to the present invention, lipid metabolism disorders, disorders characterized by lipid accumulation or weight disorders characterized by abnormal perilipin expression, or adipose tissue, serum or plasma obtained from subjects known to have Test samples can be used for diagnosis or monitoring. In one embodiment, one or more perilipin isoforms (or any combination thereof) in a test sample compared to a control sample (from a subject without lipid metabolism disorders or weight impairment) or a predetermined reference range. Is indicative of the presence of a weight disorder characterized by weight gain such as lipid metabolism disorders or obesity. In another embodiment of the present invention, an increase in the abundance of one or more perilipin isoforms (or any combination thereof) in a test sample relative to a control sample or a predetermined reference range is indicative of a cachexia Indicates the presence of a weight disorder characterized by weight loss such as quality or anorexia. In yet another embodiment, the relative abundance of one or more perilipin isoforms (or any combination thereof) in a test sample, as compared to a control sample or a predetermined reference range, is indicative of a lipid metabolism disorder, Indicates the degree or severity of disorders, diabetes or weight disorders (eg, obesity) characterized by lipid accumulation. In any of the foregoing methods, the detection of one or more of the perilipin isoforms described herein is optionally performed using one or more additional biomarkers of lipid metabolism disorders or weight disorders, such as, for example, leptin and neuropeptide Y. Can be combined with the detection of To measure the level of perilipin isoform, any suitable method in the art may be used. These include, but are not limited to, immunoassays for detection and / or visualization of perilipin isoforms (e.g., Western blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation, immunohistochemistry, Other). In addition, any suitable hybridization assay can be used to detect expression of a perilipin isoform by detection and / or visualization of mRNA encoding the perilipin isoform (eg, Northern assay, dot Blot, in situ hybridization, etc.).
[0224]
In another embodiment of the present invention, a labeled antibody that specifically binds to a perilipin isoform for the purpose of detecting, diagnosing or monitoring a weight disorder characterized by lipid metabolism disorder or abnormal perilipin expression, a derivative thereof And analogs can be used. Preferably, such disorders are detected in animals, more preferably in mammals, and most preferably in humans.
[0225]
5.10. kit
The invention also provides a kit comprising one or more agents identified by the screening assays of the invention, and instructions for use. In one embodiment, the kit comprises one or more agonists of one or more perilipin isoforms in one or more containers. In another embodiment, the kit comprises one or more antagonists of one or more perilipin isoforms in one or more containers. Preferably, the kit of the invention further comprises a control that does not function as an agonist or antagonist on the expression and / or activity of one or more perilipin isoforms.
[0226]
In one particular embodiment, the kit of the invention comprises a labeled agonist or antagonist of one or more perilipin isoforms. In a preferred embodiment, a kit of the invention comprises one or more agonists or antagonists of a perilipin isoform conjugated to a therapeutic agent. In another preferred embodiment, a kit of the invention comprises one or more agonists or antagonists of a perilipin isoform conjugated to a diagnostic agent.
[0227]
In certain embodiments, the kits of the invention include instructions for the use of antibodies for the treatment, prevention or diagnosis of a weight disorder (eg, obesity), a disorder of lipid metabolism, a disorder characterized by lipid accumulation or diabetes.
[0228]
The following examples are provided so that the invention described herein may be more fully understood. It should be understood that these examples are for illustrative purposes only and are not intended to limit the invention in any way.
[0229]
6. Example: Absence of Perilipin db / db Causes genetic low fat in mice and reverses obesity
This example demonstrates the important role of perilipin in vivo in lipid homeostasis, muscle mass and energy metabolism.
[0230]
6.1. Materials and methods
plin ^{− / −} Making and maintaining mice
A phage lambda clone was isolated from a mouse 129 genomic library using mouse perilipin cDNA obtained by PCR. This clone contained exons 1 to 7 of the perilipin gene. The replacement vector as shown in FIG. 1 was produced. This construct was introduced into mouse ES cells by electroporation (Chang et al., 1999, J. Biol. Chem. 274: 6051-6055) (R1, obtained from Dr. Andras Nagy of the University of Toronto). Eight independent recombinant ES cell clones were injected into undifferentiated germ cells from C57BL / 6J. Genotyping was performed by tail blot using XbaI restriction enzyme. All experiments were performed on F3 and F4 mice backcrossed with C57BL / 6J. Mice are weaned at 4 weeks and fed normal chow (standard Purina Rodent Chow containing 4.5% fat) or high fat (HF) feed (F-282, 35% fat from Bio-Serv, Frenchtown, NJ). , 21% protein, and 38% carbohydrate).
[0231]
Immunoblotting
Equivalent amounts of protein homogenate were separated by 4-15% SDS-PAGE, transferred to PVDF membrane, and polyclonal anti-hormone sensitive lipase (HSL) (anti-HSL antibodies were Dr. C. Holm, Lund University, Lund (Sweden)). And the anti-perilipin antibody (Research Diagnostics Inc., Flanders, NJ). Primary antibodies were visualized by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech). The relative intensity of the immunoblot bands was quantified by AlphaImager ™ 2000 Documentation & Analysis System (Alpha Innotech Corp.).
[0232]
Body composition
5 plins^{+ / +}And plin^{− / −}Male mice were sacrificed by cervical dislocation. The epididymal fat pad (pad) and gastrocnemius muscle were excised and weighed. Next, the whole carcass was homogenized with a blender. From a portion of the pre-weighed ground carcass, the fat was extracted with ethyl ether and ethyl alcohol so that the percentage of fat could be calculated from the amount of material remaining after the extraction procedure. Carcass was measured for triacylglycerol (Sigma) and protein (Bio-Rad Protein Assay).
[0233]
Glucose tolerance and insulin sensitivity test
For glucose tolerance, mice were fasted for 4 hours and then injected intraperitoneally (ip) at a dose of 3 g glucose / kg body weight. Glucose levels (FasTake, LifeScan Inc., Milpitas, Calif.) Were used to monitor glucose levels before and after injection. For insulin sensitivity, mice were fasted for 4 hours and injected intraperitoneally with 100 units / ml of normal insulin to a final concentration of 0.75 U / kg body weight. Blood was collected before injection and at 15, 30, 60 and 120 minutes after injection. Glucose was measured using a blood glucose strip.
[0234]
Blood chemistry
After the animals were anesthetized with isoflurane (Vedco, St. Joseph, MO), blood was collected from the orbital plexus. Serum was frozen in aliquots and stored at -20 ° C. Enzyme assay kits for the measurement of serum non-esterified fatty acids (NEFA C, Wako, Richmond, VA), glycerol, glucose, cholesterol, β-hydroxybutyric acid and total triacylglycerol (Sigma) were used. Serum insulin was measured using a radioimmunoassay (Linco Research, St. Charles, MO). For glucose and insulin resistance, glucose was measured by a blood glucose strip (FasTake). Corticosterone (Diagnostic Products Co., Los Angeles, Calif.) And leptin concentrations (Linco Research, St. Charles, Mo.) were measured by radioimmunoassay in plasma obtained at noon after an overnight fast.
[0235]
Hormone-sensitive lipase assay
The tissue was homogenized in 3 ml of buffer (0.25 sucrose, 1 mM EDTA, 1 mM DTE, 20 μg / ml leupeptin) per gram of tissue and centrifuged at 110,000 × g at 4 ° C. for 45 minutes. It uses a fat-depleted inflatant and is essentially described by Holm and coworkers (Holm et al., 1997, Methods Enzymol. 286: 45-67; and Osterlund et al., 1996, Biochem. J. 319: 411-420), and as a substrate the diolein analog 1 (3) -mono- [³H] oleoyl-2-O-mono-oleylglycerol (MOME) was used to measure HSL activity. One unit of enzyme activity was defined as 1 μmol of oleic acid released per minute at 37 ° C., and lipase activity was expressed as units per mg of tissue.
[0236]
Lipolysis in isolated adipocytes
Adipocytes were isolated by collagenase digestion from epididymal fat pads in the presence of adenosine as described (Rodbell, M., 1984, J. Biol. Chem. 239: 375-380). . Cells were resuspended in Kerbs-Ringer Hepes in the absence of adenosine and in the presence of 1 U / ml adenosine deaminase. 0.350x10⁶Cells / ml cells were incubated for 1 hour in the presence or absence of 2 μM CL 316,243, and extracellular glycerol release was measured as an indicator of lipolysis.
[0237]
in Vivo Lipolysis
Mice were fasted for 4 hours and given CL 316,243 (0.1 mg / kg body weight) or isoproterenol (10 mg / kg body weight) i. p. Injected. Blood was collected from the orbital plexus before injection and 15 minutes after injection, and NEFA and glycerol were measured.
[0238]
Histology
Tissues were fixed in neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin. Images were captured on a SigmaScan (Jandel, San Rafael, Calif.) And analyzed. The outline of each adipocyte was manually followed and the cytoplasmic area was measured. plin^{− / −}The size and distribution of brown adipocytes could not be accurately determined because the lipid droplets interfered with the mouse cell boundaries. The average size was estimated by dividing the total surface area by the number of nuclei.
[0239]
Measurement of oxygen consumption
Air flow rate 0.51 min^-1Oxygen consumption individually for mice fed normal diet or 35% fat diet using computer controlled open circuit indirect calorimetry at room temperature and 23 ° C. (Oxymax, Columbus Instruments Co., Columbus, OH). Was evaluated. Thirty minutes later, the mice were placed in a metabolic chamber. VO₂Was evaluated at 5 minute intervals for 20-24 hours. Mice had free access to water and food for 12 hours at night. Total oxygen consumption is expressed as the average of all samples taken during the experiment.
[0240]
Magnetic resonance imaging
Two sets of magnetic resonance imaging (MRI) experiments were performed. The first set was for determining "fat" or "lipid" images and "water" images. This is formalin-fixed plin^{− / −}And plin^{+ / +}Performed on mice. Stored specimens were securely placed inside a 25 mm NMR tube and imaged on a vertical bore, 9.4 Tesla MRI system (Varian, Palo Alto). Chemical shift selection of either water peak (4.8 ppm) or lipid peak (1.2 ppm) using a series of three 4.0 ms sinc pulses with a crusher gradient after each round. Target saturation was achieved. A 25-slice spin echo imaging pulse sequence was used. Successive 1-mm slices were selected using a 4 ms sinc pulse for excitation and refocusing. Each of the 256 phase encoder (PE) steps was acquired with a repetition time of 4 seconds, an echo time of 30 ms, and an average of 32 signals per PE step. Quantitative evaluation of% fat content in selected slices was performed at the heart and liver level. The cross-sectional area of fat measured from the fat image was divided by the total cross-sectional area of the mouse tissue in the slice determined from the water image. The cross-sectional area was quantified using image analysis software that divided the tissue according to signal intensity and counted the pixels.
[0241]
In a second set of experiments, mice were anesthetized with avertin and live plin as described in the description of FIG. 6D.^{+ / +}And plin^{− / −}For the mice, MRI spectra showing water and lipid signals were measured.
[0242]
6.2. result
Perilipin null mutant mice were produced by targeted disruption of the perilipin gene using the strategy shown in FIGS. 1A and 1B. Exon 2 and intron 2 were replaced with IRES-β-gal and neo genes in the targeting vector. Multiple targeted ES cell clones were obtained. Of the eight chimeras, five transmitted the targeted allele to progeny. Homozygous and heterozygous mice were recovered at the expected rate. Northern blotting showed no detectable perilipin mRNA (data not shown), while Western blotting showed that perilipin protein was absent in adipocytes (perilipin A and B) and testis (perilipin A and C). This was shown (FIG. 1C). plin^{− / −}The mice had no overt abnormal phenotype, were prolific and lactated normally to their offspring. When fed ad libitum, these mice had plin^{+ / +}Consumed significantly more food than littermates (plin^{− / −}Is 0.429 ± 0.054 kcal / day / g body weight, and plin^{+ / +}Is 0.332 ± 0.037 kcal / day / g body weight, p = 0.011). Plin for weight despite increased food intake^{− / −}Mouse is plin^{+ / +}There was no difference from the mice (FIGS. 2A and 2B), and their liver, kidney, spleen and heart weights were similar (data not shown). plin^{− / −}Is wild type (plin^{+ / +}) Less body fat than mice. Each fat accumulation amount is plin^{+ / +}Plin than mouse^{− / −}The mice weighed 37-38% less (FIG. 2C). However, knockout mice have their plin^{+ / +}Lower fat than mice, plin^{− / −}The total carcass lipid content of the mice was reduced by 58% (FIG. 2D) and the triacylglycerol content was reduced by 46% (FIG. 2E), while plin^{− / −}The total protein content of the mice actually increased by 21% (FIG. 2F). There was a good correlation between epididymal fat pad weight and whole body triacylglycerol content (r = 0.920, p <0.001), but body weight and fat pad weight (r = 0.199) , P = 0.582) or triacylglycerol content (r = 0.052, p <0.887). plin^{− / −}Not only was the mouse not cachectic, but in fact there was an increase in muscle mass, so that it became possible for the mouse to maintain normal body weight despite loss of body fat. plin^{− / −}The gastrocnemius muscle weight of the mouse is plin^{+ / +}7.8% higher than control (p <0.05, n = 9 for both groups, FIG. 2G).
[0243]
plin^{− / −}Adipocytes from mice are plin^{+ / +}It was substantially smaller than that of litters (FIG. 3A). plin^{+ / +}The average white adipocyte size (area) of the histological section of FIG.²And on the other hand plin^{− / −}1400 ± 48μm from mouse²The size was reduced by 62%. plin^{− / −}The much smaller white fat depot in mice was the result of a reduction in the size of individual adipocytes, not the number of adipocytes. Because the cell density is plin^{+ / +}Plin than mouse^{− / −}Is much higher (FIG. 3C), and the total DNA content of adipose tissue is^{− / −}And plin^{+ / +}(Plin^{+ / +}Plin for 140 ± 85 μg of medium^{− / −}152 ± 56 μg, n = 6 for both groups). Brown fat between shoulders is plin^{+ / +}Plin than mouse^{− / −}Medium (FIG. 3D). The average size of adipocytes in the interscapular brown adipose tissue tended to be smaller, but the differences were not significant (plin^{+ / +}3.468 ± 1.018 μg of plin^{− / −}Medium 2.607 ± 0.209 μm², P = 0.071). However, the histological appearance was very different. Because plin^{− / −}Lipid droplets in brown adipocytes are plin^{+ / +}Because it was much smaller than in brown adipocytes (FIG. 3A). plin^{− / −}The decrease in mouse total body fat reflected a corresponding decrease in its plasma leptin concentration (for male mice, plin^{+ / +}3.64 ± 1.40 ng / ml, plin for n = 7^{− / −}2.26 ± 1.23 ng / ml, n = 10, p = 0.056; and for female mice, plin^{+ / +}3.02 ± 1.40 ng / ml, plin for n = 8^{− / −}1.78 ± 0.58 ng / ml, n = 10, p = 0.001). plin^{− / −}In mice, there was no abnormal liver tissue and no evidence of intrahepatic fat infiltration (data not shown).
[0244]
plin^{− / −}Despite a large decrease in total body fat in mice, the following parameters^{− / −}And plin^{+ / +}No significant difference between the animals was detected: basal plasma cholesterol (plin^{− / −} 97 ± 18.1 mg / dl for plin^{+ / +} 89.7 ± 11.4 mg / dl) and triacylglycerol levels (plin^{− / −} Plin against 29.1 ± 6.4 mg / dl^{+ / +} 35.6 ± 6.8 mg / dl), plasma lipoprotein profile analyzed by high performance protein liquid chromatography (data not shown; Clifford et al., 2000, J. Biol. Chem. 273: 24665-24669), fasted Plasma glucose (plin^{− / −} Plin for 81.95 ± 11.24 mg / dl^{+ / +} 81.95 ± 20.03 mg / dl) and insulin (plin^{− / −} 0.206 ± 0.096 plin for ng / ml^{+ / +} 0.179 ± 0.081 ng / ml), plasma glucose response over 2 hours to an insulin tolerance test using a standard intraperitoneal (ip) insulin dose of 0.75 U / kg (data not shown), And i. p. Plasma glucose and insulin response over 2 hours to a standard glucose tolerance test using 3 g / kg glucose administered (data not shown). Plasma corticosterone levels are also plin^{− / −}(118.28 ± 54.45 ng / ml for males, n = 10 for females and 228.92 ± 56.28 ng / ml for females, n = 9) and plin^{+ / +}(108.49 ± 62.25 ng / ml for males, n = 10 and 188.55 ± 86.31 ng / ml for females, n = 10).
[0245]
The effect of a 48 hour fast on some plasma parameters is shown in FIG. 4A. There were no differences in any of the parameters at the beginning of the fast. The major differences in response to fasting were in the levels of lipolytic metabolites, non-esterified fatty acids (NEFA), and ketone bodies, β-hydroxybutyric acid. Wild-type mice were able to increase plasma NEFA and β-hydroxybutyric acid after fasting. In contrast, plin^{− / −}Although mice were able to mount a lipolytic response, they were not sufficient to increase NEFA levels. After fasting, the increase in plasma β-hydroxybutyrate was also substantially faint (FIG. 4A). plin^{− / −}Most likely, the mice were already under maximal stimulation under baseline conditions and could not increase the release of NEFA because the fat stores were much smaller (see below). It impaired the production of ketone bodies from acetyl-CoA via β-oxidation of fatty acids, since the supply of NEFAs was limited.
[0246]
Since perilipin has been reported to modulate hormone-sensitive lipase (HSL) activity, its absence appears to affect HSL activation, thereby altering the rate and energy balance of lipolysis in cells. (Londos et al., 1999, Semin. Cell Dev. Biol. 19: 51-58). According to Western blotting (FIG. 4B), plin^{+ / +}And plin^{− / −}No significant difference was detected in immunoreactive HSL between mice. Plin in subcutaneous and epididymal fat^{− / −}HSL activity in cell lysates from mice was determined to be plin^{+ / +}The corresponding fat stores of the control were 287% and 652%, respectively (FIG. 4C). plin^{− / −}HSL constitutively activated therein indicates that perilipin normally functions by controlling fat HSL activity. Reduced HSL activity in the presence of normal amounts of protein suggests that perilipin modulates HSL activity by affecting HSL access to triacylglycerols in fat droplets rather than production of total HSL. (Londos et al., 1999, Semin. Cell Dev. Biol. 19: 51-58).
[0247]
Next, the total lipolytic activity of the isolated adipocytes was measured. plin^{− / −}Basal glycerol release from isolated adipocytes of mice is determined by plin^{+ / +}Approximately 375% of those from mouse adipocytes (FIG. 4D). Addition of CL 316,243 (Muzzin et al., 1991, J. Biol. Chem. 266: 24053-24058), a specific agonist of the β-adrenergic receptor that is predominantly expressed in adipose tissue of rodents. (2 μM)^{+ / +}Stimulates glycerol release in adipocytes by about 300%, with untreated plin^{− / −}The value was almost equal to that in fat cells. In contrast, plin^{− / −}In adipocytes from mice, CL 316,243 did not cause further irritation, since lipolytic activity was already maximal under basal conditions.
[0248]
These in vitro observations prompted testing of the in vivo effects of perilipin inactivation on lipid metabolism. The lipolysis products glycerol and basal conditions and after administration of the general β-adrenergic agonists isoproterenol (10 mg / kg IP) and CL 316,243 (0.1 mg / kg IP) By measuring NEFA, plin^{− / −}And plin^{+ / +}The rate of lipolysis in mice was studied. I. Of any compound p. Blood was collected pre-dose and 15 minutes later, a time consistent with maximal plasma glycerol and NEFA responses (FIG. 4E). Before treatment, plin^{− / −}Despite much lower body fat content in mice, basal plasma glycerol and NEFA levels were reduced to plin^{− / −}And plin^{+ / +}They were almost similar to mice. After treatment with isoproterenol, plin^{+ / +}In mice, there was an approximately 800% increase in plasma glycerol levels and an approximately 300% increase in plasma NEFA levels. plin^{− / −}The corresponding increase in mice was much less pronounced, with glycerol and NEFA levels of about 300% and 0%, respectively. Similar observations were made for CL 316,243 treatment. Basal glycerol and NEFA levels were also very similar in these experiments. plin^{+ / +}In mice, CL treatment stimulated glycerol and NEFA levels by about 1100% and about 420%, respectively. plin^{− / −}In mice, stimulation with CL 316,243 was much lower, about 200% and about 140%, respectively (FIG. 4E). Differences between basal and stimulus levels are^{+ / +}Plin significantly reduced compared to control^{− / −}This can be easily explained by taking into account the adipose tissue mass of the mouse. The in vitro lipolysis experiment (FIG. 4D)^{− / −}It shows that adipocytes exhibit near maximal lipolysis under basal conditions. Thus, despite the significantly reduced fat mass, plin^{− / −}Mice were able to maintain relatively normal basal plasma glycerol and NEFA levels. Furthermore, it was confirmed in an in vivo experiment that it was observed in vitro. That is, plin^{− / −}Adipocytes are poorly stimulated by exposure to β-adrenergic agonists.
[0249]
At an ambient temperature of 21 ° C, plin^{− / −}And plin^{+ / +}Mice had similar body temperatures (FIG. 5A). plin^{− / −}Mice may have poor insulation from changes in environmental temperature due to their significantly reduced subcutaneous fat. Therefore, plin^{− / −}Mice were tested for their ability to maintain body temperature against exposure to cold. When mice were subjected to an ambient temperature of 4 ° C. in the fasted state (FIG. 5B), plin^{− / −}Mouse body temperature is plin^{+ / +}It fell much faster than that of litters, an observation consistent with poor heat preservation. However, when the experiment was repeated in the fed state (FIG. 5C), plin^{− / −}Animal is plin^{+ / +}They were able to tolerate cold as well as litters (at least 9 hours), indicating that they overcome the poor heat preservation by thermogenic response. In fact, in the feeding state, plin^{− / −}Mouse body temperature is plin^{+ / +}Tend to be slightly higher than that of food, and sufficient heat is generated in food (and oxygen) metabolism to maintain body temperature in the normal high temperature range, despite increased heat loss due to poor insulation It was shown.
[0250]
plin^{− / −}To determine if the metabolic rate of the mouse was increased, direct calorimetry^{− / −}And plin^{+ / +}The mice were tested for total oxygen consumption. When given normal food to these, plin^{− / −}Mice are slightly more (about 11%) O than wild-type animals₂Consumed (plin^{+ / +}In 11-week old mice, 42.8 ± 3.0 ml / kg / min^{− / −}47.6 ± 3.6 ml / kg / min, n = 5; p = 0.052; on an animal basis, these were plin^{+ / +}For 1.161 ± 0.074 ml / min^{− / −}Was 1.250 ± 0.179; p = 0.334). The difference was substantially greater on the high fat diet (20%) (plin^{+ / +}50.0 ± 1.9 ml / kg / min in 11 week old mice, plin for n = 6^{− / −}59.2 ± 3.2 ml / kg / min, n = 4; p <0.001; on an animal basis, these were plin^{+ / +}For 1.340 ± 0.088 ml / min^{− / −}1.486 ± 0.077 ml / min; p = 0.024). plin^{− / −}And plin^{+ / +}O between mouse₂The difference in consumption was greater when the mice were awake and eating than when they were asleep (FIG. 5D).
[0251]
After eating a high fat (35%) diet for 3.5 months, plin^{+ / +}The mice became obese, while plin^{− / −}The mice were relatively resistant to diet-induced obesity (FIGS. 6A and 6B). Under these conditions, wild-type mice developed a large epididymal fat pad. plin^{− / −}The size of the mouse epididymal fat pad also increased, but to a relative weight not different from that observed in wild-type animals on a normal diet (FIG. 6A). Furthermore, after feeding a high fat diet, the total body (carcass) fat content was plin^{+ / +}Plin compared to mice^{− / −}Almost 50% lower in mice (plin^{− / −}At 21.7%, plin^{+ / +}41.4%, FIG. 6B).
[0252]
Inactivation of the perilipin gene also protected db / db mice (Chen et al., 1996, Cell 84: 491-495), a genetic model of obesity due to leptin resistance, from the development of obesity. By crossing two types of animals, plin^{− / −}Db / db mice in which the allele was inherited were obtained. Notably, these mice had lost the obese phenotype (FIGS. 6C and 6D). The protective effect of perilipin's absence was evident early in life. At 6 weeks, wild-type mice weigh 17.59 ± 2.59 g (n = 11), db / db / plin^{+ / +}Weighs 33.45 ± 1.61 g (n = 4) and db / db / plin^{− / −}Was 24.90 + 2.16 g (n = 3). db / db / plin^{− / −}And db / db / plin^{+ / +}The significant difference in weight between and continued as the mice grew. At week 12 db / db / plin weighing 30.2 g^{− / −}Wild type (n = 11) and db / db / plin compared to mice^{+ / +}(N = 5) weighed 24.93 ± 1.37 g and 49.83 ± 4.14 g, respectively. At 20 weeks, the weight was 28.78 + 2.14 g for the wild type (n = 11), db / db / plin^{+ / +}60.75 + 2.09 g for (n = 5), and db / db / plin^{− / −}33.5 g for the mouse. As shown in FIGS. 7A and 7B, db / db / plin^{− / −} The weight of the mouse is this db / db / plin^{− / −}As mice age, they approach the weight of wild-type mice. Thus, knockout of the perilipin allele in db / db mice at least partially reverses the obesity phenotype associated with the db / db genotype. Further, db / db / plin^{− / −}Mouse is db / db / plin^{+ / +}A delayed onset of diabetes was presented compared to mice.
[0253]
Using magnetic resonance imaging (MRI), plin^{− / −}Mouse and plin^{+ / +}The fat area of the control cross section was quantified. Fat image is plin^{+ / +}In animals, it was about 15%^{− / −}Showed a total cross-sectional area of approximately 7.6% through the heart region. In slices of the liver region, the fat area is plin^{+ / +}About 9.24% of^{− / −}Then it was about 3.5%. Thus, the reduction in fat area of individual sections was 50-65% (data not shown). However, when using whole body MRI,^{− / −}Total lipid content (FIG. 6D, top right panel) is plin^{+ / +}The decrease was about 36% compared to the mouse (FIG. 6D, upper left panel). db / db / plin^{+ / +}And db / db / plin^{− / −}Similar analysis of mice shows that db / db subcutaneous and visceral fat is significantly reduced and total lipid signal is reduced to db / db / plin.^{+ / +}From about 63% to db / db / plin^{− / −}It became clear that the value was reduced to about 27%, which was a value close to that of the wild type (about 24%) (FIG. 6D, lower panel). These MRI analyzes confirm that inactivation of perilipin in mice reverses the obese phenotype of db / db mice. Excess body fat loss in double knockout mice was associated with increased metabolic rate, as assessed by oxygen consumption. Depending on whether the mouse measures while eating or sleeping, VO₂Was apparent (FIG. 5E).
[0254]
6.3. Consideration
Applicant is plin^{− / −}Mice were created, which are healthy, muscular and low-fat. These mice are resistant to diet-induced obesity, and plin in db / db mice^{− / −}Allelic inheritance reverses the obesity phenotype. plin^{− / −}The obesity-resistant phenotype in mice is the result of its high metabolic rate. One of the mechanisms for increased energy expenditure in these mice is that they have a leaner body, which is metabolically active. Another possible mechanism is that the free cycle of lipogenesis and lipolysis, which can re-esterify free fatty acids produced in adipocytes in situ and consume ATP, leading to increased oxygen consumption. There is something.
[0255]
plin^{− / −}Mice are very different from the obesity-resistant Hmgic-deficient mice with the recently described dwarf phenotype (Zhou et al., 1995, Nature 376: 771-774; Andand et al., 2000, Nat. Genet. 24: 377). -380; Hirning-Folz et al., 1998, Genes Chrom. Cancer 23: 350-357; and Benson et al., 1994, Genet. Res. 64: 27-33). Adipocytes are one of many mesenchymal tissues affected by Hmgic deficiency. Hmgic> 50% lighter than wild-type littermates^{− / −}Unlike mice (Anand et al., 2000, Nat. Genet. 24: 377-380), plin^{− / −}Mice have normal body weight and increased muscle mass despite reduced fat content.
[0256]
plin^{− / −}The mice were mice in which acyl CoA: diacylglycerol transferase was inactivated (Dgat; Smith et al., 2000, Nature Genet. 25: 87-90) and those in which protein kinase A RIIβ subunit was inactivated (Cummings et al. 1996, Nature 382: 622-626). All three types of mice exhibit increased energy expenditure and constitutive low fat (Smith et al., 2000, Nature Genet. 25: 87-90; and Cummings et al., 1996, Nature 382: 622-626). The mechanism of manifestation of the phenotype of Dgat inactivation, including defects in normal milk production, is unclear (Smith et al., 2000, Nature Genet. 25: 87-90). Inactivation of the protein kinase A RIIβ subunit stimulates lipolysis, apparently as a result of a compensatory increase in the protein kinase A RIα subunit (Cummings et al., 1996, Nature 382: 622-626). The protein kinase A RIIβ subunit is expressed in adipose tissue and brain, and is also expressed at low levels elsewhere. One possible interpretation of the data from mice with inactivated protein kinase A RIIβ subunits is that the low-fat phenotype of RIIβ inactivation is more likely than the inactivation of fat RIIβ to “ Directly from neuronal changes "(Planas et al., 1999, J. Biol. Chem. 274: 36281-36287). In contrast, perilipin is expressed exclusively in adipose tissue and at very low levels in steroidogenic tissues. Its inactivation in adipocytes appears to be directly responsible for increased lipolytic activity, increased basal metabolic rate, and constitutively low fat.
[0257]
7. Example: Absence of Perilipin ob / ob Reverse obesity in mice
This example demonstrates the important role perilipin plays in lipids and energy metabolism in vivo. This example also demonstrates a beneficial effect that is the result of down-regulation of perilipin expression or activity.
[0258]
7.1. Materials and methods
ob / ob / plin ^{− / −} mouse
ob / ob mouse and plin^{− / −}By crossing mice, db / db / plin^{− / −}Mice were made.
[0259]
Northern blot analysis
RNA was isolated from various tissues by the Ultraspec RNA isolation kit from Biotecz, Houston, Texas. The hybridization probe used was³²The corresponding mouse cDNA was P-labeled and cloned. RNA (20 μg) was electrophoresed on a 1% agarose gel and transferred to a Hybond-N nylon membrane filter. Corresponding filter³²Hybridized with a P-labeled cDNA probe (see FIGS. 10-12) at 42 ° C. for 16 hours, then sequentially with 2 × SSC, 0.05% SDS and 2 × SSC, 0.1% SDS, and finally 0.1 × SDS. Washed with SSC, 0.1% SDS at 65 ° C. for 30 minutes (1 × SSC = 15 mM sodium citrate, 150 mM sodium chloride, pH 9.0). The filters were exposed to X-ray film with two intensifying screens at -80 ° C for 24-72 hours.
[0260]
Glucose tolerance test
For glucose tolerance, mice were fasted for 10 hours and then injected intraperitoneally (ip) at a dose of 1.5 g glucose / kg body weight. Glucose levels (FasTake, LifeScan Inc., Milpitas, CA) were used to monitor glucose levels before and after injection. Crystal Chem Inc. Insulin levels before and after injection were monitored using an ELISA kit from (Chicago, ILL).
[0261]
7.2. result
Inactivation of the perilipin gene protected ob / ob mice, a genetic model of obesity, from the development of obesity. The protective effect of perilipin's absence was evident early in life. As shown in FIGS. 8A and 8B, ob / ob / plin^{− / −}The weight of the mouse is ob / ob / plin^{+ / +}It decreased compared to the weight of the mouse. ob / ob / plin^{− / −}The weight of the mouse is this ob / ob / plin^{− /-}As mice age, they approach the weight of wild-type mice. Thus, knockout of the perilipin allele in ob / ob mice at least partially reverses the obesity phenotype associated with the ob / ob genotype.
[0262]
Next, a Northern blot assay was performed to determine the effect of perilipin inactivation on expression of enzymes involved in β-oxidation. FIG. 9 shows the steps of β-oxidation. As shown in FIGS. 10 and 11, increased mRNA levels for major β-oxidase were detected primarily in adipose tissue, heart and muscle. These results suggest that one of the mechanisms that leads to fat degradation from perilipin inactivation is due to activated β-oxidation. One valid hypothesis is plin^{− / −}In mice, the synthesis of triacylglycerols is unchanged or only slightly altered, and even if triacylglycerols are synthesized, they are immediately degraded by lipolysis and waste ATP. That is, an idling cycle is performed.
[0263]
Because UCPs are known to reduce energy consumption efficiency by producing thermal energy by “wasting ATP”, perilipin inactivation in response to stimulation of uncoupled protein (UCP) activity The impact was evaluated. There are three highly homologous UCPs, UCP-1, UCP-2 and UCP-3, and a few others with substantially lower homology. As shown in FIG. 12, when Northern blot analysis was used, there was no difference in the concentration of UCP-1 mRNA expression in brown adipose tissue, but in brown and white adipose tissue and the heart, UCP-2 mRNA was expressed. An increase in concentration was detected. These results indicate that plin with respect to increased metabolic rate and oxygen consumption^{− / −}This suggests that one of the possible mechanisms of the mouse is the induction of UCPs.
[0264]
The effect of perilipin inactivation on glucose intolerance (obese diabetes mellitus) in ob / ob mice was evaluated. Glucose and insulin concentrations from plasma of ob / ob mice with or without wild-type and perilipin alleles were measured after a 10 hour fast. 13A and 13B graphically show fasting plasma glucose and fasting plasma insulin concentrations detected in mice, respectively. As shown in FIG. 14A, i. p. Blood glucose levels after administration showed a diabetic curve. In contrast, ob / ob / plin^{− / −}The blood glucose levels of the mice were not indistinguishable from wild-type controls, but were not significantly different. As shown in FIG. 14B, i. p. Blood insulin levels after administration increased compared to wild-type controls. Inactivation of the perilipin allele was determined by glucose i.p. p. Thirty minutes after administration, blood insulin levels were reduced in ob / ob mice (FIGS. 14A and 14B). These results suggest that in ob / ob mice, inactivation of perilipin reverses glucose intolerance and reverses abnormal insulin secretion. There are two mechanisms for the beneficial effects of perilipin down-regulation: (i) reducing insulin resistance in ob / ob mice by reducing the amount of body fat; and (ii) secretion of insulin To improve.
[0265]
The present invention should not be limited in scope by the exemplary embodiments intended to illustrate one aspect of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[0266]
All of the publications cited herein are incorporated by reference in their entirety.
[Brief description of the drawings]
FIG.
plin^{− / −}1 shows the production of mice. A. Targeting strategy. B.サ Southern blotting of tail DNA after XbaI digestion. C.ウ ェ ス タ ン Western blotting of cell extracts from epididymis and subcutaneous fat (left) and testis (right).
FIG. 2
Perilipin inactivation is plin^{− / −}Figure 2 shows phenotypic effects on body weight, fat accumulation, lipid content, and muscle mass in mice.
FIG. 3
Figure 4 shows the effect of perilipin inactivation on adipose tissue.
FIG. 4
A. @Plin^{+ / +}And plin^{− / −}Figure 4 shows the effect of a 48 hour fast on body weight and plasma parameters in mice. B.ウ ェ ス タ ン Shows Western blotting of HSL extracted from epididymal fat. C. Ｈ HSL activity of isolated adipocytes. D.示 す Shows the lipolysis rate in the separated fat cells. E. FIG. @Plin^{+ / +}And plin^{− / −}Figure 4 shows lipolysis in mice.
FIG. 5
A. ＰPlin kept at 21 ℃^{+ / +}And plin^{− / −}1 shows the body temperature of a mouse. B.体 Shows the body temperature of mice exposed to an ambient temperature of 4 ° C. without feeding. C.体 Shows the body temperature of mice exposed to an ambient temperature of 4 ° C. with food. D. $ Plin fed diet containing 35% fat^{+ / +}And plin^{− / −}Representative oxygen consumption (VO) from indirect calorimetry in mice₂) Indicates tracing. E. FIG. １４ 14 week old db / db / plin fed normal diet^{+ / +}And db / db / plin^{− / −}Representative VO in mice₂Show tracing.
FIG. 6
A. Ｐ plin after 3.5 months of regular diet or 35% fat (HF) diet^{+ / +}And plin^{− / −}4 shows the epididymal fat pad amount of a group of mice. B. Ｐ plin after 3.5 months of regular diet or 35% fat (HF) diet^{+ / +}And plin^{− / −}3 shows the total carcass fat content of mice. C. Db / db / plin^{+ / +}, Db / db / plin^{− / −}, Plin^{+ / +}And plin^{− / −}A photograph of the mouse is shown. D.示 す Magnetic resonance imaging (MRI) of body fat distribution.
FIG. 7
A.雄 Male db / db (triangle) over 4-17 weeks, plin^{+ / +}(Squares), and db / db / plin^{− / −}(Circle) shows the weight of the mouse. B.雌 Female db / db (triangles), plin over 4-22 weeks^{+ / +}(Squares), and db / db / plin^{− / −}(Circle) shows the weight of the mouse.
FIG. 8
A.雄 Male db / db (squares), plin over 6-27 weeks^{+ / +}(Diamond), and db / db / plin^{− / −}(Triangle) Indicates the weight of the mouse. B.雌 Female db / db (square), plin over 6-24 weeks^{+ / +}(Diamond), and db / db / plin^{− / −}(Triangle) Indicates the weight of the mouse.
FIG. 9
3 shows the steps of β-oxidation.
FIG. 10
Figure 2 shows expression of fatty acyl-CoA dehydrogenase.
FIG. 11
3 shows expression of trifunctional proteins.
FIG.
Figure 2 shows the expression of uncoupling protein (UCP).
FIG. 13
A.示 す Show fasting plasma glucose levels. B.を Show fasting plasma insulin levels.
FIG. 14
3 shows a glucose intolerance test.

Claims (31)

A method of identifying a drug to be tested for its ability to regulate weight, body fat or muscle mass, comprising:
(A) contacting a perilipin isoform or fragment thereof with a candidate agent for a time sufficient to form a perilipin isoform or fragment / drug complex;
(B) measuring the level of a perilipin isoform or fragment / drug complex;
That, if the measured level differs from the level measured in the absence of the candidate drug, the agent to be tested for its ability to modulate body weight, body fat or muscle mass has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate the development, development or progression of diabetes, comprising:
(A) contacting a perilipin isoform or fragment thereof with a candidate agent for a time sufficient to form a perilipin isoform or fragment / drug complex;
(B) measuring the level of a perilipin isoform or fragment / drug complex;
That, if the measured level differs from the level measured in the absence of the candidate drug, that the drug to be tested for its ability to modulate the development, development or progression of diabetes has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate lipid metabolism, comprising:
(A) contacting a perilipin isoform or fragment thereof with a candidate agent for a time sufficient to form a perilipin isoform or fragment / drug complex;
(B) measuring the level of a perilipin isoform or fragment / drug complex;
The method, wherein if the measured level differs from the level measured in the absence of the candidate agent, the agent to be tested for its ability to modulate lipid metabolism has been identified. A method of identifying a drug to be tested for its ability to modulate weight, body fat or muscle mass, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent for a time sufficient to form a perilipin isoform / drug complex;
(B) measuring the level of the perilipin isoform / drug complex;
That, if the measured level differs from the level measured in the absence of the candidate drug, the agent to be tested for its ability to modulate body weight, body fat or muscle mass has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate the development, development or progression of diabetes, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent for a time sufficient to form a perilipin isoform / drug complex;
(B) measuring the level of the perilipin isoform / drug complex;
That, if the measured level differs from the level measured in the absence of the candidate drug, that the drug to be tested for its ability to modulate the development, development or progression of diabetes has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate lipid metabolism, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent for a time sufficient to form a perilipin isoform / drug complex;
(B) measuring the level of the perilipin isoform / drug complex;
The method, wherein if the measured level differs from the level measured in the absence of the candidate agent, the agent to be tested for its ability to modulate lipid metabolism has been identified. A method of identifying a drug to be tested for its ability to regulate weight, body fat or muscle mass, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent;
(B) measuring the induced level of cellular second messengers or the phosphorylation level of perilipin isoforms;
The result is that if the measured level differs from the level measured in the absence of the candidate agent, the compound to be tested for the ability to modulate body weight, body fat or muscle mass has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate the development, development or progression of diabetes, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent;
(B) measuring the induced level of cellular second messengers or the phosphorylation level of perilipin isoforms;
If the level measured differs from the level measured in the absence of the candidate agent, then the compound to be tested for its ability to modulate the development, development or progression of diabetes has been identified. The above method. A method of identifying an agent to be tested for its ability to modulate lipid metabolism, comprising:
(A) contacting a population of cells expressing a perilipin isoform with a candidate agent;
(B) measuring the induced level of cellular second messengers or the phosphorylation level of perilipin isoforms;
The method, wherein if the measured level differs from the level measured in the absence of the candidate agent, the compound to be tested for its ability to modulate lipid metabolism has been identified. 8. The method of claim 1, 4 or 7, wherein the ability to adjust body weight is the ability to lose weight. 8. The method of claim 1, 4 or 7, wherein said ability to adjust body weight is the ability to increase body weight. 8. The method of claim 1, 4 or 7, wherein the ability to regulate body fat is the ability to reduce body fat. 9. The ability to modulate the onset, development or progression of diabetes, wherein the ability to lower blood glucose levels, decrease insulin sensitivity, or suppress one or more signs or symptoms associated with diabetes. The method described in. The method of claim 1, 4 or 7, wherein the ability to regulate muscle mass is the ability to increase muscle mass. 10. The method of claim 3, 6, or 9, wherein the ability to regulate lipid metabolism is the ability to decrease lipid metabolism. 10. The method of claim 3, 6 or 9, wherein the ability to modulate lipid metabolism is the ability to increase lipid metabolism. A method of identifying a drug that modulates animal weight or body fat, comprising:
(A) modulating the expression of one or more perilipin isoforms, or modulating one or more activities of one or more perilipin isoforms, in an animal or group of animals that binds to one or more perilipin isoforms; Administering a candidate drug to
(B) determining whether the candidate agent modulates body weight or body fat in an untreated control animal or group of animals compared to an untreated control animal or group of animals;
The method, wherein if the candidate agent modulates body weight or body fat, an agent that modulates the weight or body fat of the animal has been identified. A method of identifying a drug that modulates muscle mass in an animal, comprising:
(A) modulating the expression of one or more perilipin isoforms, or modulating one or more activities of one or more perilipin isoforms, in an animal or group of animals that binds to one or more perilipin isoforms; Administering a candidate drug to
(B) determining whether the candidate agent modulates muscle mass in an untreated control animal or group of animals as compared to an untreated control animal or group of animals;
The method, wherein if the candidate agent modulates muscle mass, the agent that modulates muscle mass in the animal has been identified. A method of identifying an agent that modulates the development, development or progression of diabetes in an animal, comprising:
(A) modulating the expression of one or more perilipin isoforms, or modulating one or more activities of one or more perilipin isoforms, in an animal or group of animals that binds to one or more perilipin isoforms; Administering a candidate drug to
(B) determining whether the candidate agent modulates blood glucose, insulin sensitivity, or one or more signs or symptoms of diabetes in the untreated control animal or group of animals compared to an untreated control animal or group of animals.
Comprising identifying a drug that modulates the development, development or progression of diabetes in an animal if the candidate drug modulates blood glucose, insulin sensitivity, or one or more signs or symptoms of diabetes. The above method. A method of identifying an agent that modulates lipid metabolism in an animal, comprising:
(A) modulating the expression of one or more perilipin isoforms, or modulating one or more activities of one or more perilipin isoforms, in an animal or group of animals that binds to one or more perilipin isoforms; Administering a candidate drug to
(B) determining whether the candidate agent modulates lipid metabolism in the untreated control animal or group of animals compared to an untreated control animal or group of animals.
The method, wherein if the candidate agent modulates lipid metabolism, the agent that modulates lipid metabolism in the animal has been identified. 21. The method of claim 17, 18, 19, or 20, wherein the animal or group of animals is a livestock, poultry, or companion animal. 21. The method of claim 17, 18, 19, or 20, wherein the animal or group of animals is a human. A method of preventing or treating a disorder characterized by weight gain in a subject, comprising administering to a subject in need or desire of such treatment a therapeutically effective amount of one or more of the perilipin expression or activity antagonists. A method of administering a compound of the formula (I). A method of preventing or treating a disorder characterized by weight loss in a subject, comprising administering to a subject in need of or desired such treatment a therapeutically effective amount of one or more agonists of perilipin expression or activity. A method of administering a compound of the formula (I). A method of preventing or treating diabetes in a subject, comprising administering to a subject in need or desire for such treatment a therapeutically effective amount of one or more compounds that function as antagonists of perilipin expression or activity. A method comprising: 24. The method of claim 23, wherein said weight disorder is obesity. 25. The method of claim 24, wherein the disorder is cachexia or anorexia. A method of increasing lipid metabolism and increasing muscle mass in a subject comprising administering to a subject in need of or desirous such treatment a therapeutically effective amount of one or more compounds that function as antagonists of perilipin expression or activity. A method comprising administering. A method of increasing lipid accumulation in a subject, comprising administering to a subject in need or desire for such treatment a therapeutically effective amount of one or more compounds that function as agonists of perilipin expression or activity. The method comprising. 29. The method of claim 23, 24, 25, 27 or 28, wherein said subject is a human. 29. The method of claim 23, 24, 25, 27 or 28, wherein the subject is a livestock or poultry.

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