JP2003528804A - Methods and compositions for controlling bone formation through modulation of leptin activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for treating, diagnosing and preventing bone diseases, comprising a method comprising leptin synthesis, leptin receptor synthesis, leptin binding to leptin receptor and inhibition or increase of leptin receptor activity. The present invention also relates to screening assays for identifying compounds that modulate leptin and / or leptin receptor activity. The invention further relates to gene therapy utilizing leptin and leptin-related sequences for the treatment and prevention of bone diseases.

Description

Detailed Description of the Invention

This application is US provisional patent application No. 60 / 138,733 (1999) under 35 U.SC Article 119 (e).
It claims priority based on the application dated June 11, 2012). The present invention is based on the US Natio
completed with government support under grant numbers NIHR01DE11290, NIHR01AR45548 and NIHR01AR43655 awarded by the National Institute of Health, the Government has certain rights.

[0002] The present invention is not limited, for example conditions associated with bone, including osteoporosis, disorders, the treatment of diseases, related to diagnosis and prevention. Furthermore, the invention relates to the regulation of the receptor signaling pathway of the polypeptide hormone leptin. The present invention particularly relates to regulation of leptin synthesis, leptin receptor synthesis, binding of leptin to the receptor, and regulation of leptin which is a signal to bone cells. The invention further provides methods for the identification, prophylactic and therapeutic use of compounds in the treatment, prognosis and diagnosis of bone-related conditions, disorders, diseases. Furthermore, the present invention provides for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of bone-related conditions, disorders, for monitoring the efficacy of compounds in clinical subjects, as well as bone-related conditions, disorders. , Provides a method for identifying humans susceptible to disease.

[0003] Physiological processes reconstruction of background bone invention is carried out by constantly updating the bone via two distinct continuous cellular processes (Karsenty, 1999, Genes and Develop
ment, 13; 3037-3051). The first event is the resorption of preexisting bone by osteoclasts, and then the formation of new bone by osteoclasts. The two processes in bone remodeling must maintain the balance of bone mass within the narrow limits between the end of puberty and gonad arrest. The molecular mechanism responsible for maintaining this constant bone mass is unknown, but some evidence suggests that, at least in part, involves complex endocrine rules to achieve this. I am trying. For example, gonadal dysfunction and the resulting lack of sex steroids
It stimulates the bone resorption process of bone remodeling, ultimately leading to osteopenia (low bone mass) or osteoporosis (low bone mass and easy fracture). Similarly, recent identification of osteoprotegerin in serum and its functional characterization via its body tissue route further suggests that secreted molecules influence osteoclast bone resorption. (Simonet, et al., 1997, Cell, 89: 309-319). This systemic control of bone resorption suggests that other as yet unspecified circulating molecules may regulate bone formation via osteoblasts. The identification of these hormones and growth factors, if present, is of great importance to the onset and prevalence of certain diseases that affect bone remodeling.

One of these diseases is osteoporosis (Riggs et al., 1998, JB.
one Miner. Res., 13: 763-773). Osteoporosis is the most common symptom affecting bone remodeling and is the most common disease in the western hemisphere. At the physiopathological level, the hallmark of this disease is a reduction in bone mass, ie, low density and prone to fracture. Moreover, the onset of osteoporosis in both men and women is closely associated with gonad function arrest and is less common in obese individuals. At the cellular level, osteoporosis is a loss of balance of bone remodeling where bone resorption to bone formation is prioritized, which results in reduced bone mass and increased fracture. At the molecular level, the etiology of osteoporosis is largely unknown.

[0005] SUMMARY OF THE INVENTION An object of the invention is the treatment of bone disease via modulation of the receptor signal pathway, it is to perform a diagnosis and / or prophylaxis. Bone diseases that can be treated and / or prevented by the present invention include diseases characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. For example, but not limited to, osteoporosis, osteopenia, osteoporosis and the like. Bone disorders that can be treated and / or prevented by the present invention further include disorders characterized by increased bone mass relative to that of the corresponding non-diseased bone. For example, but not limited to, osteopetrosis
, Osteosclerosis, osteomalacia, etc.

According to one aspect of the present invention, a method of treating a bone disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that reduces serum leptin levels. And a bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. A specific example of such a compound and method is to use one that inhibits or reduces leptin synthesis or increases leptin destruction. Such compounds include, but are not limited to, the triple-helical sequences of antisense, ribozyme or leptin encoding polypeptides. According to one aspect of the invention, a method of treating a bone disorder comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that reduces leptin levels in cerebrospinal fluid. , In which the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that inhibit or reduce leptin synthesis or increase leptin destruction, or compounds that bind leptin in blood.

In a particular embodiment of the method of the present invention, a method of treating a bone disorder comprising administering a therapeutically effective amount of a compound to a mammal in need thereof. A bone disease is characterized by a decrease in bone mass as compared to that of a corresponding non-diseased bone, wherein the compound binds leptin in the blood, such as, but not limited to, specifically binds leptin. An antibody, a soluble leptin receptor polypeptide,
Inter α-trypsin-inhibitor heavy chain-related protein, and alpha 2
-Selected from the group consisting of macroglobulin proteins.

According to another aspect of the invention, a method of treating a bone disorder comprising phosphorylated Sta.
administering a therapeutically effective amount of a compound that reduces the level of t3 polypeptide to a mammal in need of treatment of a bone disorder, the reduction of bone mass relative to that of the non-diseased bone to which the bone disorder corresponds. A method is provided that is characterized. Specific examples of such compounds and methods include, but are not limited to, those that inhibit or decrease leptin synthesis or increase leptin destruction, compounds that bind leptin in blood, and the use of leptin receptor antagonists. Therefore, examples of this leptin receptor antagonist are selected from antibodies that specifically bind to leptin and antibodies that specifically bind to leptin receptor. Further, a compound containing a soluble leptin receptor / polypeptide sequence can also be used.

According to another aspect of the invention, a method of treating a bone disorder, wherein a therapeutically effective amount of a compound that reduces leptin receptor levels in the hypothalamus is administered to a mammal in need of treatment for the bone disorder. Administration, wherein the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that inhibit or decrease leptin receptor synthesis or increase leptin receptor destruction. Such compounds include, but are not limited to, the triple helix sequences of polypeptides encoding antisense, ribozyme or leptin receptors. According to another aspect of the invention, a method of treating a bone disorder, wherein a therapeutically effective amount of a compound that increases leptin levels in serum and / or spinal fluid is administered to a mammal in need of treatment for the bone disorder. In this case, a method is provided in which the bone disease is characterized by an increase in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin receptor synthesis or decrease leptin destruction.

According to another aspect of the invention, a method of treating a bone disease comprising phosphorylated Sta.
comprising administering to a mammal in need of treatment of a bone disorder a therapeutically effective amount of a compound that increases the level of t3 polypeptide, wherein the bone disorder is of bone mass relative to that of the corresponding non-diseased bone. Methods are provided that are characterized by augmentation. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin synthesis or decrease leptin destruction, and further use leptin receptor antagonist compounds.

According to another aspect of the present invention, there is provided a method of treating bone disease, wherein a therapeutically effective amount of a compound that increases leptin receptor levels in the hypothalamus is administered to a mammal in need of treatment of bone disease. Administering, wherein the bone disease is characterized by an increase in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin receptor synthesis or decrease leptin receptor destruction.

According to another aspect of the present invention, there is provided a method of preventing bone disease, which comprises providing a compound that reduces serum leptin levels to prevent bone disease in a mammal at risk of bone disease. Administration at a sufficient concentration consists in preventing bone disease, which is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to,
The use is of one that suppresses or reduces leptin receptor synthesis or increases leptin destruction. Such compounds include, but are not limited to, the triple-helical sequences of antisense, ribozyme or leptin encoding polypeptides. According to another aspect of the present invention, a method of preventing bone disease, wherein the compound that reduces leptin levels in cerebrospinal fluid is sufficient to prevent bone disease in a mammal at risk of bone disease Administration at a concentration comprises preventing bone disease, which is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to,
The use of a compound that inhibits or reduces leptin synthesis, or increases destruction of leptin, or a compound that binds to leptin in blood.

A particular embodiment of the method of the present invention includes a method of preventing bone disease, which method comprises providing the compound at a concentration sufficient to prevent bone disease in a mammal at risk for bone disease. A method of preventing a bone disease by administration in which the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone, wherein the compound binds leptin in the blood A compound selected from compounds such as, but not limited to, an antibody that specifically binds leptin, a soluble leptin receptor polypeptide, an inter-alpha-trypsin-inhibitor heavy chain-related protein, and alpha.
2-selected from the group consisting of macroglobulin proteins.

According to another aspect of the invention, a method of preventing bone disease comprising phosphorylated Sta.
Preventing bone disease by administering to a mammal at risk of bone disease a compound that reduces the level of t3 polypeptide in a concentration sufficient to prevent bone disease, wherein said bone disease is Is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that inhibit or decrease leptin synthesis or increase leptin destruction, compounds that bind leptin in blood, and the use of leptin receptor antagonists. Thus, examples of this leptin receptor antagonist include an acetylphenol compound, an antibody that specifically binds to leptin, an antibody that specifically binds to leptin receptor, and a compound containing a soluble leptin receptor / polypeptide sequence. Is also included.

According to another aspect of the invention, a method of preventing bone disease comprising administering to a mammal at risk of bone disease a compound that reduces leptin receptor levels in the hypothalamus. In which case the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that inhibit or decrease leptin receptor synthesis or increase leptin destruction. Such compounds include
Included, but not limited to, the triple helix sequences of polypeptides encoding antisense, ribozyme or leptin receptors. According to another aspect of the present invention, a method of preventing bone disease comprising a compound that increases leptin levels in serum and / or cerebrospinal fluid to prevent bone disease in a mammal at risk of bone disease. At a concentration sufficient to provide a method wherein the bone disease is characterized by an increase in bone mass relative to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin synthesis or decrease leptin destruction.

According to another aspect of the present invention, there is provided a method of preventing bone disease comprising phosphorylated Sta.
Preventing bone disease by administering to a mammal at risk of bone disease a compound that increases the level of t3 polypeptide in a concentration sufficient to prevent bone disease, wherein the bone disease is Is characterized by an increase in bone mass relative to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin synthesis or decrease leptin destruction, and the use of leptin receptor agonist compounds.

According to another aspect of the present invention, a method of preventing bone disease comprising preventing a bone disease in a mammal at risk of bone disease by a compound that increases leptin receptor levels in the hypothalamus. At a concentration sufficient to do so, wherein the bone disease is characterized by an increase in bone mass as compared to that of the corresponding non-diseased bone. Specific examples of such compounds and methods include, but are not limited to, those that increase or induce leptin receptor synthesis or decrease leptin receptor destruction.

According to yet another aspect of the invention there is provided a method of diagnosing or prognosing a bone disease in a mammal, eg a human, which method comprises: (a) suffering from a mammal, eg a bone disease, Or measuring the leptin level in serum of a mammal at risk thereof; (b) comparing the leptin level measured in (a) above with the leptin level in control serum; measured in (a) above. The level is higher than that in the control serum, the mammal is diagnosed or predicted to have, or be at risk for, bone disease.
In this case, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of diagnosing or prognosing a bone disease in a mammal, eg a human, which method comprises: (a) suffering from a mammal, eg a bone disease, Or measuring the leptin level in cerebrospinal fluid of a mammal at risk thereof; (b) comparing the level measured in (a) above with the leptin level in control cerebrospinal fluid; measuring in (a) above If the level produced is higher than that in the control cerebrospinal fluid, then the mammal has, or is at risk of being diagnosed with, bone disease.
In this case, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of diagnosing or prognosing a bone disease in a mammal, eg a human, which method comprises: (a) having a mammal, eg a bone disease, Or measuring the leptin level in serum of a mammal at risk thereof; (b) comparing the leptin level measured in (a) above with the leptin level in control serum; measured in (a) above. The level is lower than that in the control serum, the mammal is diagnosed as having or at risk for a bone disease, in which case the bone disease is compared to that of the corresponding non-diseased bone. And is characterized by increased bone mass.

According to yet another aspect of the invention there is provided a method of diagnosing or prognosing bone disease in a mammal, eg a human, which method comprises: (a) having a mammal, eg a bone disease, Or measuring the leptin level in cerebrospinal fluid of a mammal at risk thereof; (b) comparing the level measured in (a) above with the leptin level in control cerebrospinal fluid; measuring in (a) above If the level produced is lower than that in the control cerebrospinal fluid, then the mammal has, or is at risk of being diagnosed with, bone disease.
In this case, the bone disease is characterized by an increase in bone mass as compared to the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, eg a human, which method comprises (a) (B) measuring the leptin level in the serum of the mammal; (c) comparing the level measured in (b) above to the leptin level in the serum of the mammal prior to administration of the compound. Thereby monitoring the potency of the compound, in which the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, eg a human, the method comprising: (B) the leptin level in the cerebrospinal fluid of the mammal is measured; (c) the level measured in (b) above is the leptin level in the cerebrospinal fluid of the mammal before the administration of the compound. , Which monitors the potency of the compound, in which bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, eg a human, the method comprising: (B) measuring the leptin level in the serum of the mammal; (c) comparing the level measured in (b) above to the leptin level in the serum of the mammal prior to administration of the compound. Thereby monitoring the potency of the compound, in which bone disease is characterized by increased bone mass as compared to that of the corresponding non-diseased bone.

According to yet another aspect of the invention there is provided a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, eg a human, which method comprises (a) (B) the leptin level in the cerebrospinal fluid of the mammal is measured; (c) the level measured in (b) above is the leptin level in the cerebrospinal fluid of the mammal before the administration of the compound. , Which monitors the potency of the compound, in which bone disease is characterized by an increase in bone mass compared to that of the corresponding non-diseased bone.

According to yet another aspect of the invention, there is provided a method for identifying a compound for testing the ability to regulate bone mass in a mammal, which method comprises: (a) contacting the test compound with a polypeptide. (B) It is determined whether or not the test compound binds to the polypeptide, and when the test compound binds to the polypeptide, the test compound is recognized for its ability to regulate bone mass. The polypeptide is selected from the group consisting of a leptin polypeptide and a leptin receptor / polypeptide.

According to yet another aspect of the invention, there is provided a method for identifying a compound that regulates bone mass in a mammal, the method comprising: (a) contacting a test compound with a polypeptide; b) identifying a test compound bound to the polypeptide; (c) administering the test compound of (b) above to a non-human mammal and determining whether the test compound regulates bone mass in the mammal. Is measured in the context of the corresponding bone of an untreated control non-human mammal; the polypeptide is selected from the group consisting of a leptin polypeptide, and a leptin receptor polypeptide. When the compound regulates bone mass, it is characterized by recognizing the presence of a compound that regulates bone mass in a mammal.

According to yet another aspect of the invention, a method for identifying a compound that modulates (increases or decreases) mammalian bone mass comprising: (a) a test compound as a leptin polypeptide and leptin. The receptor / polypeptide is contacted for a time sufficient to form a leptin / leptin receptor complex; (b) the leptin / leptin receptor complex level is measured; It is characterized by recognizing the presence of a test compound having the function of regulating bone mass when it differs from the level measured in the absence of the compound.

According to yet another aspect of the invention there is provided a method for identifying a compound to be tested for its ability to reduce bone mass in a mammal comprising: (a) expressing a functional leptin receptor with the test compound. (B) measuring whether or not the test compound activates the leptin receptor, and when the test compound activates the leptin receptor, a compound having the ability to reduce bone mass It is characterized by recognizing its presence in mammals.

According to yet another aspect of the invention there is provided a method for identifying a mammalian bone mass reducing compound comprising: (a) contacting a test compound with a cell expressing a functional leptin receptor. Then, it is determined whether or not the test compound activated the leptin receptor; (b) administration of the test compound recognized as activating the leptin receptor in (a) above to a non-human animal And determine whether the test compound reduces bone mass in an animal in relation to the corresponding bone in a non-human control animal; if the test compound reduces bone mass in the animal, bone mass Is characterized by recognizing the presence in the mammal of a compound that reduces the.

According to yet another aspect of the invention, a method for identifying a compound to be tested for its ability to increase bone mass in a mammal comprising: (a) a leptin polypeptide and a test compound being a functional leptin. Contacting with cells expressing the receptor; (b) determining whether the test compound reduced activation of the leptin receptor in the context of what was observed in the absence of the test compound; When the test compound reduces the activation of the leptin receptor, the compound recognizes its presence in mammals as a compound having the ability to increase bone mass.

According to yet another aspect of the present invention there is provided a method for identifying a compound that increases mammalian bone mass comprising: (a) expressing a leptin polypeptide and a test compound as a functional leptin receptor. To determine whether or not the test compound reduces the activation of the leptin receptor, and (b) the test recognized as reducing the activation of the leptin receptor in (a) above. The compound is administered to a non-human animal and it is determined whether the test compound increases the bone mass of the animal in the context of the corresponding bone of a non-human control animal; Recognizing the presence of a compound that increases bone mass in a mammal when the amount is increased.

According to yet another aspect of the invention there is provided a method for identifying a compound that increases bone mass in a mammal comprising: (a) expressing a leptin polypeptide and a test compound as a functional leptin receptor. To determine whether or not the test compound reduces the activation of the leptin receptor, and (b) the test recognized as reducing the activation of the leptin receptor in (a) above. The compound is administered to a non-human animal and it is determined whether the test compound increases the bone mass of the animal in the context of the corresponding bone of a non-human control animal; Is characterized by recognizing the presence in the mammal of a compound that increases bone mass.

The present invention further provides pharmaceutical compositions used to treat and / or prevent bone disorders.

Other objects, features and advantages will be easily understood from the following specification, reference to the accompanying drawings forming a part thereof, and description of preferred embodiments. In addition,
These examples are merely for purposes of disclosure as preferred examples.

3.1 Definitions The following terms are used under the following definitions.

Leptin (used herein as “Ob”) is defined as the endogenous polypeptide product of the ob gene, preferably the human ob gene, whose known activity is via the hypothalamus. Mediated.

A leptin receptor (herein used as “ObR”) is a receptor through which the leptin hormone binds and produces a signal, preferably the term It refers to the human leptin receptor.

Bone disease, as used herein, refers to any bone disease or condition that results in, or is characterized by, loss of health or loss of bone integrity. But not osteoporosis, osteopenia, incomplete bone formation,
Includes osteoblasts, fractures, bone tumors, osteopetrosis, osteosclerosis, osteomalacia. In particular, bone diseases that can be treated and / or prevented by the present invention include bone diseases characterized by a decrease in bone mass in comparison with the corresponding non-diseased bone (e.g. osteoporosis, osteopenia,
Osteoarthritis), and bone diseases characterized by increased bone mass in comparison to corresponding non-diseased bone (eg, osteopetrosis, osteosclerosis, osteomalacia). Bone disease prevention includes actively interfering with disease prevention before it becomes ill. For the treatment of bone diseases, after the onset of the disease, it actively interferes with the activity of the disease or medical condition, and improves the symptoms.
Includes reversing. In particular, treatment herein refers to a method of adjusting bone mass to a condition very close to that of a corresponding non-diseased bone in a non-diseased condition (eg, corresponding bone of the same type as that of long spinal cord bone, etc.). It is a thing.

As used herein, a leptin receptor antagonist refers to a factor that neutralizes, prevents, or otherwise reduces the action, effect of a leptin receptor. Such antagonists include compounds that bind to leptin or bind to leptin receptors. This antagonist neutralizes and interferes with leptin receptor output,
Further included is a compound that otherwise reduces, the leptin receptor output is an intracellular step in the leptin signaling pathway after leptin binds to the leptin receptor, that is, it affects the leptin / leptin receptor signal. A downstream event,
This does not occur at the receptor / ligand interaction level. This leptin receptor antagonist includes, but is not limited to, proteins, antibodies, small organic molecules,
Carbohydrates such as acetylphenol compounds, antibodies that specifically bind to leptin, antibodies that specifically bind to leptin receptor, compounds having a soluble leptin receptor polypeptide sequence are included.

As used herein, a leptin receptor agonist refers to a factor that activates, induces, interferes with, or otherwise increases the action or effect of a leptin receptor. Such agonists include compounds that bind leptin or bind leptin receptors. The antagonists further include compounds that activate, induce, or otherwise increase leptin receptor output, which is the leptin signaling pathway after leptin binds to the leptin receptor. An intracellular process, ie a downstream event affecting the leptin / leptin receptor signal, which does not occur at the receptor / ligand interaction level. The leptin receptor agonist specifically binds to, but is not limited to, proteins, antibodies, small organic molecules, carbohydrates such as leptin, leptin analogs, leptin,
Antibodies that activate are included.

The drug is administered in a “therapeutically effective amount”, which is the amount that produces the desired change in the physiology of the mammal to which it is administered, ie, in the context of the corresponding bone of the disease state. To increase or decrease bone mass, which results in treatment of corresponding non-diseased bone that is not in a diseased state (eg, corresponding bone of the same type as that of long spinal bone) , Means to get closer.

[0043]   Here, ECD refers to the extracellular domain.

[0044]   Here, TM refers to a transmembrane domain.

[0045]   Here, CD refers to the cytoplasmic domain.

4. Brief Description of the Drawings (See below for a description of the drawings) 5. DETAILED DESCRIPTION OF THE INVENTION Various aspects of the invention are described in detail below.

5.1 Leptin and Leptin Receptor Proteins, Polypeptides, Nucleic Acids Leptin (“Ob”) and leptin receptor (“ObR”) proteins and nucleic acids (sense and antisense) are therapeutic, diagnostic, It can be used as part of a prognostic and testing method. For example, an Ob and / or ObR protein,
Polypeptides and peptide fragments, mutated, truncated or deleted forms of Ob or ObR, eg, soluble derivatives such as, but not limited to, peptides or polypeptides corresponding to one or more leptin receptor ECDs Notched leptin receptor polypeptides lacking one or more ECDs or TMs, leptin and leptin receptor fusion protein products (leptin receptor Ig fusion protein, ie fusion of leptin receptor to IgFc domain, or leptin The domain of the receptor) can be used.

Leptin and leptin receptors are known, including human leptin and leptin receptors. The leptin receptor protein is disclosed in the following documents. That is, Friedman and Halaas, 1998,
Nature, 395: 763-770; U.S. Pat. 5,972,621. The leptin sequence is disclosed in the following documents, including the human leptin coding sequence and the leptin gene regulatory sequence. That is, Zhang, Y
． , Other, 1994, Nature, 372: 425-432; de la.
Browse et al., 1996, PNAS93: 4096-4101; He.
Others, 1995, J. Biol. Chem. 270: 28887-28891
Hwang et al., 1996, PNAS 93: 873-877; and Gon;
g. Other, 1996, J.I. Biol. Chem. 271: 3971-3974 and the like.

For example, one or more peptides and polypeptides corresponding to ObR (eg, ECD, TM or CD), truncated or deleted Ob or ObR (eg, ObR deficient in TM and / or CD). , And full-length Ob or ObR, Ob
A fusion protein in which an ObR peptide or a truncated Ob or ObR (eg, ObR ECD, TM or CD domain) is fused to a heterologous or unrelated protein is also included in the scope of the present invention, and a known Ob or ObR nucleotide and Ob or ObR amino acid are known. It can be used or designed based on the arrangement. Preferably, the leptin polypeptide is capable of binding the leptin receptor under standard physiological and / or cell culture conditions. Similarly, preferably, the leptin receptor polypeptide is capable of binding leptin under standard physiological and / or cell culture conditions. That is, at least the leptin receptor polypeptide comprises a sufficient leptin amino acid sequence for leptin receptor binding, ie, for leptin / leptin receptor complex formation. Similarly, at least the leptin receptor polypeptide is one that comprises sufficient leptin receptor ECD sequences for leptin binding.

With respect to ObR peptides, polypeptides, fusion peptides and fusion polypeptides comprising all or part of the ObR ECD, such peptides include soluble leptin receptor polypeptides. Preferably, such soluble leptin receptor polypeptides are capable of binding leptin under standard physiological and / or cell culture conditions. Thus, at a minimum, such soluble leptin receptor polypeptides will contain sufficient ObR ECD sequences for leptin binding.

The fusion protein includes, but is not limited to, an IgFc fusion that stabilizes a soluble ObR protein or peptide and prolongs half-life in vivo;
Or a fusion to any amino acid sequence capable of immobilizing a fusion protein on a cell membrane and exposing ECD on the cell surface; or an enzyme, a fluorescent protein or a enzyme that provides a marker or reporter function useful for the examination and / or diagnosis of the present invention Fusions to photoproteins are included.

Ob or ObR polypeptides and peptides can be chemically synthesized (eg, Creighton, 1983, Proteins: Structures).
and Molecular Principles, W.M. H. Freeman
& Co. , N .; Y. ). You can The Ob and ObR encoding polynucleotides refer to the upstream and downstream sequences within the Ob and ObR genes as well as the sequences that encode the open reading frame. This method can be used to construct expression vectors containing Ob and ObR polynucleotide sequences. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in v
Including ivo gene recombination (Sambrook, et al., 1989, Molec
ural Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, N.M. Y. , And Au
sabel, et al., 1989, Current Protocols in
Molecular Biology, Green Publishing
Associates and Wiley Interscience, N.M.
Y. ,checking. ) Additionally, RNA capable of encoding Ob and ObR polynucleotide sequences can also be chemically synthesized using a synthesizer (eg,
"Oligonucleotide Synthesis", 1984, Gai
t, M .; J. ed. , IRL Press, Oxford).

A variety of host expression vector systems may be utilized to express the Ob and ObR polynucleotide sequences of the present invention. If the Ob and ObR peptides or polypeptides are soluble derivatives (eg, ObR peptides corresponding to ECD; TM and / or CD deleted notches or deleted ObR), the peptides or polypeptides are from the culture, ie , The ObR peptide or polypeptide is not secreted, and from the culture medium when the ObR peptide or polypeptide is secreted by the cell. However, this expression system also includes treated host cells expressing Ob and ObR or functional equivalents in the field of use, ie those anchored in the cell membrane. Purification and enrichment of Ob or ObR from such an expression system can be carried out using an appropriate detergent (detergent), lipid micelle and a known method. However, use of this treated host cell itself when it is important to maintain the structural and functional properties of Ob and ObR and to assess biological activity, eg in drug testing. You can

This expression system used for the purposes of the present invention includes, but is not limited to, bacteria modified with recombinant bactericidal viral DNA, plasmid DNA or cosmid DNA expression vectors containing Ob and ObR nucleotide sequences. (For example, microorganisms such as E. coli, B. subtilis); yeast modified with a recombinant yeast expression vector containing the above nucleotide sequence (Saccharomyce).
s, Pichia); a recombinant viral expression vector (eg, b
insect cell system infected with A. aculovirus; recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV
A plant cell system infected with a tobacco mosaic virus (TMV) or modified with a recombinant plasmid expression vector (eg, Ti plasmid) containing the above nucleotide sequence; a genome of mammalian cells (eg, metallothionein promoter) or mammals; Mammalian cell systems harboring recombinant expression constructs having promoters derived from viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter) (eg, COS, CH
O, BHK, 293, 3T3) and the like.

In the bacterial system, many expression vectors may be advantageously selected depending on the intended use for the Ob or ObR gene product expressed. For example, for the production of a pharmaceutical composition of Ob or ObR protein, or Ob or Ob
If it is desired to produce large quantities of the protein to produce antibodies to the R protein, vectors that give high levels of expression of the fusion protein product, which are easy to purify, are preferred. Examples of such vectors include, but are not limited to, E
． coli expression vector pUR278 (Ruther, others, EMBOJ.2
: 1791) in which the Ob or ObR coding sequences are individually linked to the vector in frame with the lacZ coding region, thereby producing a fusion protein; pIN vector (Inouye & Inouye, 198).
5, Nucleic Acids Res. 13: 3101-3109; Van
Heeke & Schuster, 1989, J. Am. Biol. Chem. 264
: 5503-5509) and the like. The pGEX vector is also glutathione S
-It can be used to express an external polypeptide as a fusion protein with transferase (GST). Generally, such fusion proteins are soluble and can easily be purified from degraded cells by adsorption to glutathione-agarose beads and elution in the presence of free glutathione. This pGEX vector was designed to contain a thrombin or Factor Xa protease cleavage site, thus allowing the cloned target gene product to be released from the GST moiety.

In the insect system, Autographa califonica nucleia polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is Spodopter Frugiperda
a frugiperda) cells are grown. The Ob or ObR gene coding sequences are individually cloned into a nonessential region of the virus (eg polyhedrin gene) and placed under control of an AcNPV promoter (eg polyhedrin promoter). The proper insertion of the Ob or ObR gene coding sequences inactivates the polyhedrin gene and produces an undisturbed recombinant virus (ie, a virus lacking the protein coating encoded by the polyhedrin gene). It These recombinant viruses were followed by Spodoptera
It is used to infect Frugiperda cells, in which the inserted gene is expressed (eg Smith, et al., 1983, J. Virol. 46: 584; S
Smith, US Patent No. 4,215,051).

For mammalian host cells, a number of viral-based expression systems are available. When adenovirus is used as an expression vector, the relevant O
The b or ObR nucleotide sequence binds to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence.
This chimeric gene is then inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, the E1 or E3 region) renders the recombinant virus variable and allows expression of the Ob or ObR gene product in an uninfected host (eg, , Logan & Shenk, 1984, Proc. Natl. Acad. S.
ci. USA 81: 3655-3659). Specific initiation signals will be required for efficient translation of this inserted Ob or ObR nucleotide sequence. These signals include the ATG start codon and adjacent sequences. Ob or O
If the bR gene or cDNA, including its own initiation codon and adjacent sequences, were all inserted into a suitable expression vector, then additional translational control signals would not be required. However, if only part of the coding sequence is inserted, exogenous translational control signals will likely be required, including the ATG start codon. In addition, the initiation codon should be synchronized with the reading frame of the desired coding sequence,
You need to ensure that the entire insert is translated. There are various sources of these exogenous translational control signals and initiation codons such as natural ones and artificial ones. The efficiency of expression can be improved by including appropriate transcription enhancing elements, transcription terminators, etc. (Bittner, et al., 1987, Met.
hods in Enzymol. 153: 516-544).

In addition, the host cell line may be chosen appropriately to control the expression of the inserted sequences or to modify or engineer the gene product in the specific fashion desired. Improvements (eg glycosylation) and processing (eg cleavage) of protein products are important for protein function. Different host cells have unique mechanisms and features for post-translational processes and improvements in protein and gene products. Appropriate cell lines or host systems should be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells may be used which have the cellular machinery for the correct processing of primary transcription, glycosylation and phosphorylation of the gene product. Examples of such mammalian host cells include CHO, VERO, BHK, HeLa, COS, MDCK, 293, 313,
W138, in particular, but not limited to choroidal cell lines.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines can be created that stably express the Ob or ObR sequences. Instead of using an expression vector containing a replicating viral source, the host cell with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. Can be transformed. After the introduction of external DNA, the treated cells are allowed to grow in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid is resistant to selection, allowing the plasmid to integrate into the cell's chromosome and grow to form foci. It will be cloned instead and expanded into a cell line. This method is Ob
Alternatively, it can be advantageously used to generate cell lines that express the ObR gene product. This constructed cell line is particularly useful for the examination and evaluation of compounds that affect the endogenous activity of Ob or ObR gene products.

Many selection systems can be used. For example, without limitation, herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell,
11: 223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Szybalski, 1962, Proc. Natl.
． Acad. Sci. USA, 48: 2026) and adenine phosphoribosyl transferase (Lowy, et al., 1980, Cell, 22: 817).
The gene can be used in tk ', hgprt', aprt 'cells, respectively. Antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, which is resistant to methotrexate (Wig.
Ler, et al., 1980, Natl. Acad. Sci. USA, 77:35.
67; O'Hare, et al., 1981, Proc. Natl. Acad. Sc
i. USA, 78: 1527); gpt, this gene is resistant to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. A.
cad. Sci. USA, 78: 2072); neo, which is resistant to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150: 1); and hygro, which is hygromycin. Shows resistance to (Santerre and others, 1
984, Gene 30: 147).

The Ob or ObR gene product can also be expressed in transgenic animals. Any animal, including but not limited to mice, rats, guinea pigs, pigs, micropigs, goats, non-human primates such as baboons, monkeys, chimpanzees, can be used to generate transgene animals.

Known techniques can also be used to introduce an Ob or ObR transgene into an animal or to knock out or inactivate an endogenous Ob or ObR to produce a founder line of transgene animals. Such animals can be used as part of the inspection method of the present invention. The cells and / or tissues of these animals can be obtained for development of another composition (eg, cell line, tissue culture system),
This can be used as part of the inspection method of the present invention.

Techniques for the development of such animals are known, for example pronuclear microinjection (Hoppe, PC and Wagner, TE, 1989,
US Patent No. 4,873,191); retrovirus-mediated gene transcription into the germ line (Van der Putten, et al., Proc. Natl. Aca.
d. Sci. USA, 82: 6148-6152); targeting genes to embryonic stem cells (Thompson, et al., 1989, Cell, 56: 313-321).
; Embryo electroporation (Lo, 1
983, Mol, Cell. Biol. 3: 1803-1814); sperm-mediated gene transcription (Lavitrano, et al., 1989, Cell, 57: 717-.
723). As a reference for such a technique, Gorgon, 1989, Trans
genetic Animals, Intl. Rev. Cytol. 115: 171
-229 can be mentioned.

Regarding transcribed animals containing the transgene Ob and / or ObR, such animals carry the Ob and / or ObR transgene in all of their cells. Alternatively, such animals, like mosaic animals, carry this transgene in part, but not in all cells. The transgene is integrated as a single transgene or as a chain, eg, head-to-head tandem or head-to-tail tandem. This transgene can be selectively introduced or activated into specific cell types as follows (Lasko, M,
Others, 1992, Proc. Natl. Acad. Sci. USA, 89: 6
232-6236). The regulatory sequences required for the specific activation of such cell types will depend on the particular cell type of interest, and will be apparent to those of skill in the art. Gene targeting is preferred when it is desired to integrate the transgene into the chromosomal site of the endogenous gene. Briefly, if one wishes to utilize such a technique, a vector having a nucleotide sequence similar to the endogenous Ob or ObR gene is designed for coalescence. That is, they are integrated into the nucleotide sequence of the endogenous Ob or ObR gene by disrupting its function through similar recombination by chromosomal sequences. Transgenes can also be selectively introduced into specific cell types. This causes the transgene to inactivate the endogenous Ob or ObR gene only in that cell type. After that, it is processed by the method of Gu et al. (Gu, et al., 1994, Science, 265: 103-1).
06). The regulatory sequences required for the specific activation of such cell types will depend on the particular cell type of interest, and will be apparent to those of skill in the art.

Once the transgene animal has developed, recombinant gene expression is assessed using standard techniques. Initial test is by Southern blot analysis or PCR
The technique is used to analyze animal tissues to assess whether transgene integration has taken place. Transgene mRNA in the tissues of animals
The level of expression is also assessed by specific techniques. Techniques in this case include, but are not limited to, Northern blot analysis of tissue samples obtained from animals, in situ hybridization analysis, and RT-PCR. Ob and Ob
Samples of R gene expressing tissues are also evaluated immunocytochemically using specific antibodies for transgene products.

5.1.1 Antibodies to Ob and ObR Proteins Specifically recognizes and binds to one or more epitopes of Ob or ObR, or epitopes of conserved variants of Ob or ObR, or peptide fragments of Ob or ObR. Antibodies can be used as part of the method of the invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (
ab ') ₂ fragments, fragments obtained from Fab expression libraries, anti-idiotype (anti-Id) antibodies and epitope binding fragments of any of the above.

Such antibodies are for example diagnostic or of the invention for diagnosing bone disorders in a mammal by measuring leptin levels in the mammal (eg leptin levels in the serum or cerebrospinal fluid of the mammal). It can be used as part of a prognosis method. Such antibodies can also be utilized in compound screening schemes, such as those set forth below, to assess the effect of a test compound on the expression and / or activity of the Ob or ObR gene product, for example. Moreover, such antibodies can be used in the therapeutic and prophylactic methods of the invention. For example, such an antibody may correspond to a leptin receptor agonist or antagonist.
In addition, such antibodies can be administered to reduce brain leptin levels, as assayed by leptin levels in cerebrospinal fluid. further,
Such antibodies can be used to reduce leptin levels by increasing the rate at which leptin is cleared from the circulation (e.g., can promote leptin degradation), or such antibodies can be used to reduce leptin reception. By increasing the rate at which the body (and cells expressing the leptin receptor) degrades or disintegrates, leptin receptor levels can be reduced, including reduction of cells expressing the leptin receptor.

To generate antibodies, Ob or ObR, Ob or ObR peptides (for ObR, for example, those which correspond to the functional domain of the receptor, eg ECD, TM or CD)
, A truncated Ob or ObR polypeptide (eg, for ObR, wherein one or more domains such as TM or CD are deleted), a functionally equivalent Ob or ObR, or By injecting a variant of Ob or ObR,
A variety of host animals can be immunized. Such host animals include, but are not limited to, rabbits, mice, and rats, just to name a few. Various adjuvants can be used to enhance the immune response, depending on the host species. It includes Freund (complete and incomplete), inorganic gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, surfactants such as peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG (Bacille). Calmette-Guerin)
And potentially useful human adjuvants such as Corynebacterium parvum, but are not limited thereto. Polyclonal antibodies are a heterogeneous group of antibody molecules obtained from the sera of immunized animals.

Monoclonal antibodies are homogeneous populations of antibodies directed against a particular antigen.
It can be obtained by any technique that results in the production of antibody molecules by continuous culture cell lines. These are the hybridoma methods of Kohler and Milstein (1975
, Nature 256: 495-497; and U.S. Pat. No. 4,376,110), human B cell hybridoma method (Kosbor et al., 1983, Immunology Today 4: 72; Cole et al., 1983, Proc. Natl.
Acad. Sci. USA 80: 2026-2030), and the EBV-hybridoma method (Cole et al., 19
85, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77
-96), but is not limited to these. Such antibodies include IgG, IgM, IgE
, IgA, IgD and any subclass thereof, including any immunoglobulin class. The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. The above is the preferred method of production as it produces high titers of mAbs in vivo.

Furthermore, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, that comprise both human and non-human portions can be made using standard recombinant DNA techniques. , Within the scope of the invention. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as molecules with variable regions and human immunoglobulin constant regions from mouse mAbs (eg, Cabilly et al., US Pat. No. 4,816,567; and Boss et al. , U.S. Pat. No. 4,816,397, which is hereby incorporated by reference in its entirety). Humanized antibodies are antibody molecules from non-human species that have one or more complementarity determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, eg, Queen, US Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies may be incorporated into groups known in the art. It can be produced by a modified DNA technique, for example PCT WO 87/02671; European Patent Application No. 171,496;
European Patent Application No. 173,494; PCT WO 86/01533 Publication; US Patent No. 4,816,567;
European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (19).
87) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. Immunol. 139.
: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimu.
ra et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985). Sc
ience 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; US Pat. No. 5,225,5.
39; Jones et al. (1986) Nature 321: 552-525; Verboeyan et al. (1988) Science 239:
1534; and Beidler et al. (1988) J. Immunol. 141: 4053-5060.

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be made, for example, using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but capable of expressing human heavy and light chain genes. Transgenic mice are immunized in the usual manner with a selected antigen, eg, all or part of a polypeptide of the invention. Monoclonal antibodies against the above-mentioned antigens can be obtained using a conventional hybridoma method. Human immunoglobulin transgenes introduced into transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, therapeutically effective Ig using the method as described above
G, IgA and IgE antibodies can be made. For a review of the above techniques for making human antibodies, see Lonberg and Huszer (1995, Int. Rev. Immunol. 13: 65.
-93). For a detailed discussion of the above techniques for making human and human monoclonal antibodies, and protocols for making such antibodies, see, eg, US Pat. No. 5,625,126; US Pat. No. 5,633,425, US Pat. See 5,569,825, US Pat. No. 5,661,016, US Pat. No. 5,545,806. In addition, it is possible to contract with a company such as Abgenix, Inc. (Fremont, CA) to receive human antibodies against selected antigens using methods similar to those described above.

Fully human antibodies that recognize selected epitopes can be made using the method called "induced selection." In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope. (Lespers et al. (1994) Bio / technology 12: 8
99-903).

Alternatively, the technique described for the production of single chain antibodies (US Pat. No. 4,946,778)
Issue; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad.
Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-546), Ob.
And can be applied to generate single chain antibodies to the ObR gene product. Single chain antibodies are made by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes are produced by known methods. For example, such fragments are F (ab ') ₂ fragments generated by cleaving antibody molecules with pepsin, and Fab fragments that can be generated by reducing the disulfide bridges of the F (ab') ₂ fragments. But is not limited to. We also constructed a Fab expression library (Hus
e et al., 1989, Science), allowing rapid and convenient identification of monoclonal Fab fragments with the desired specificity.

Antibodies to Ob or ObR can also be used to generate anti-idiotypic antibodies "mimicking" Ob or ObR using methods known to those of skill in the art. (For example, Greenspan & Bona, 1993. FASEB J 7 (5): 437-444; and Nissinof, 1991.
, J. Immunol. 147 (8): 2429-2438). For example, antibodies that bind to the ObR ECD and competitively inhibit the binding of Ob to ObR can be used to generate anti-idiotypes that mimic ECD, thereby binding and neutralizing Ob. it can. Such neutralizing anti-idiotypes, or Fab fragments of such idiotypes, for neutralizing Ob and treating bone disorders characterized by decreased bone mass as compared to corresponding non-diseased bone Can be used in therapy.

5.2 Diagnosis and Prognosis of Bone Diseases and Compound / Patient Monitoring Various methods for diagnosing bone diseases or conditions and assessing prognosis, and for identifying subjects who are predisposed to such diseases or conditions. Any method can be used. The above bone diseases or conditions include osteoporosis, osteopenia, incomplete ossification or resorption, osteoblastitis, bone fracture, bone metastasis, osteopetrosis, osteosclerosis and osteochondrosis. However, it is not limited to these.

In particular, bone disorders that can be diagnosed or predicted in accordance with the present invention include bone disorders characterized by decreased bone mass as compared to the corresponding non-diseased bone, which further comprises
Includes, but is not limited to, osteoporosis, osteopenia and osteoarthritis.

Thus, according to the above aspects of the invention, there is a method for diagnosing or prognosing bone disease in a mammal, such as a human, which is (a) indicative of the mammal, eg bone disease. Measuring the leptin concentration in the serum of a mammal, or a mammal suspected of being at risk, and (b) comparing the concentration measured in (a) with the leptin concentration in the control serum. It consists of If the resulting concentration in (a) is higher than the control, then the mammal is diagnosed with or at risk for bone disease. Here, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone.

There are also methods for diagnosing or prognosing bone disease in mammals such as humans, which include (a) mammals, eg, mammals that exhibit or are at risk of bone disease. Determining the leptin concentration in the cerebrospinal fluid of the animal; and (b) comparing the concentration measured in (a) with the leptin concentration in the control cerebrospinal fluid. If the resulting concentration in (a) is higher than the control, then the mammal is diagnosed with or at risk for bone disease. Here, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone.

Further, bone disorders that can be diagnosed or predicted in accordance with the present invention include bone disorders characterized by increased bone mass as compared to the corresponding non-diseased bone, which further includes osteopetrosis. , Osteosclerosis and osteochondrosis, but is not limited thereto.

Thus, according to the above aspects of the invention, there is a method for diagnosing or prognosing bone disease in a mammal, such as a human, which is (a) indicative of the mammal, eg bone disease. Measuring the leptin concentration in the serum of a mammal, or a mammal suspected of being at risk, and (b) comparing the concentration measured in (a) with the leptin concentration in the control serum. It consists of If the resulting concentration in (a) is lower than the control, then the mammal is diagnosed with or at risk for bone disease. Here, the bone disease is characterized by an increase in bone mass as compared to the corresponding non-diseased bone.

There are also methods for diagnosing or prognosing bone disease in mammals such as humans, which include: Determining the leptin concentration in the cerebrospinal fluid of the animal; and (b) comparing the concentration measured in (a) with the leptin concentration in the control cerebrospinal fluid. If the resulting concentration in (a) is lower than the control, then the mammal is diagnosed with or at risk for bone disease. Here, the bone disease is characterized by an increase in bone mass as compared to the corresponding non-diseased bone.

Further provided are methods for diagnostically monitoring patients undergoing clinical evaluation for the treatment of bone disease, and for assessing the efficacy of the compounds in clinical trials.

Thus, in yet another aspect of the invention, there is a method for investigating the efficacy of a compound for treating a bone disease in a mammal, such as a human, which comprises (a) Administration of the compound to a mammal; (b) measuring the leptin concentration in the serum of the mammal; and (c) the concentration measured in (b) of the mammal prior to administration of the compound. Comparing with leptin concentration in serum, thereby examining the efficacy of this compound. Here, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone. Desirable compounds are compounds that increase leptin levels relative to the levels observed prior to administration.

In yet another aspect of the invention, there is a method for investigating the efficacy of a compound for treating a bone disease in a mammal, such as a human, which comprises: (a) (B) measuring the leptin concentration in the cerebrospinal fluid of the mammal; and (c) the concentration measured in (b) in the cerebrospinal fluid of the mammal before the administration of the compound. , Compared to the leptin concentration of, to study the potency of this compound. Here, the bone disease is characterized by a decrease in bone mass as compared to the corresponding non-diseased bone. Desirable compounds are compounds that increase leptin levels relative to the levels observed prior to administration.

According to yet another aspect of the invention is a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, such as a human, which method comprises: (a) administering the compound to the mammal. Administering; (b) measuring serum leptin levels in the mammal; and (c) comparing the levels measured in (b) to serum leptin levels in the mammal prior to administering the compound, This monitors the potency of the compound, wherein the bone disease is characterized by increased bone mass relative to the bone mass of the corresponding non-diseased bone. Preferred compounds are those that reduce leptin levels relative to the levels observed prior to administration.

According to another aspect of the invention is a method of monitoring the efficacy of a compound for treating a bone disorder in a mammal, such as a human, which method comprises: (a) administering the compound to the mammal. (B) measuring the level of cerebrospinal fluid leptin in the mammal; and (c) comparing the level measured in (b) with the level of cerebrospinal fluid leptin in the mammal prior to administration of the compound. This monitors the potency of the compound, wherein the bone disease is characterized by an increased bone mass relative to the bone mass of the corresponding non-diseased bone. Preferred compounds are those that reduce leptin levels relative to the levels observed prior to administration.

Methods such as these can also be used to monitor patients undergoing clinical evaluation for the treatment of bone disorders. Generally, such methods involve monitoring bone mass for corresponding non-diseased bone.

The methods described herein include Ob and ObR nucleotide sequences known to those of skill in the art as described above, for example, as described in Section 5.1.1 (eg, US Pat.
5,972,621), and reagents such as the Ob and ObR antibodies. Ob is typically expressed in adipocytes and lower levels are also found in stomach and lymphocytes. ObR is typically expressed in the hypothalamus in the brain. Friedman Halaas, 1998, Nature, 395: 763-770. Thus, these reagents can be used, for example: (1) to detect the presence of Ob and ObR gene mutations, or to Ob or ObR mRNA for non-bone disease states, eg in mammalian serum or cerebrospinal fluid.
(2) detection of over or under-abundance of the Ob or ObR gene product for non-bone disease states in, for example, serum or in mammalian serum; and (3) mediated by Ob or ObR It can be used to detect perturbations or abnormalities in the signal transduction pathway. Alternatively, the phosphorylation level of Stat3 protein
It can be measured against the corresponding control sample or the level observed in the mammal. Stat3 phosphorylation is a biochemical event that occurs after leptin binds to the leptin receptor. Devos et al., 1997, JBC, 272: 18304-18310.

The methods described herein can be performed in conjunction with these techniques, either before or after the techniques for measuring bone mass. For example, in identifying a mammal (eg, a human) that exhibits high or low leptin levels (eg, in serum or cerebrospinal fluid) relative to that of a comparable control sample, the bone mass of that individual is measured,
It can be further elucidated whether the mammal exhibits increased or decreased bone mass relative to the corresponding non-diseased bone. If no abnormal bone mass is observed, the mammal can be considered at risk of developing the disease, whereas if abnormal bone mass is observed, the mammal is indicative of bone disease.

Techniques well known to those of skill in the art for measuring bone mass include, among others, skeletal X-rays that show the transparency of bone (the lower the transparency, the higher the bone mass); classical bone histology (eg relative to controls and / Or bone volume to osteoclasts, trabecular / number and aspect of trabecular formation, number of osteoblasts); and bone mass, dual energy X-ray absorptiometry commonly used for osteoporosis ( DEXA) (Levis and Altman, 1998, Arthritis a
nd Rheumatism, 41: 577-587), but is not limited thereto.

The methods described herein can further be used to diagnose individuals at risk for bone disease. Such individuals include menopausal women (as used herein, the term refers to about 6 months before menopause begins to about 1 postmenopausal).
(Includes a time frame of up to 8 months), and patients undergoing treatment with corticosteroids, especially long-term corticosteroids, but are not limited thereto.

The methods described herein may include, for example, at least one specific Ob or O 2
Pre-packaging diagnostic kits containing bR nucleotide sequences or Ob or ObR antibody reagents, which can be conveniently used, for example, in clinical settings to diagnose patients with bone disease.

Any nucleated cell can be used as a starting source for genomic nucleic acid to detect the Ob or ObR mutations. To detect Ob or ObR gene expression or gene product, it is possible to utilize any cell type or tissue in which the Ob or ObR gene is expressed, such as the vascular cells for ObR. it can.

Nucleic acid based detection techniques are described below in Section 5.2.1. Peptide detection techniques are described below in Section 5.2.2.

5.2.1 Detection of Ob and ObR Genes and Transcripts Mutations in Ob and ObR can be detected using a number of techniques. Nucleic acid from any nucleated cell can be used as a starting point for such assay techniques and can be isolated according to standard nucleic acid preparation procedures well known to those of skill in the art.

DNA can be used in hybridization and amplification assays of biological samples to detect abnormalities associated with the Ob or ObR gene structure, including point mutations, deletions and chromosomal translocations. Such assays include, but are not limited to, Southern analysis, single-stranded conformational polymorphism analysis (SSCP) and PCR analysis.

Such diagnostic methods for detecting Ob and ObR gene-specific mutations can be performed, for example, by recombinant DNA molecules obtained from a sample, eg derived from a patient sample or other suitable cell source, cloned Nucleic acid containing a gene or degenerate variants thereof is used to specifically bind a recombinant DNA molecule, a cloned gene or a nucleic acid reagent containing a degenerate variant thereof, to these complementary sequences in the Ob or ObR gene, respectively. This involves contacting and incubating under conditions that favor annealing. Preferably, the length of these nucleic acid reagents is at least 152-30 nucleotides. After incubation, all unannealed nucleic acid is removed from the nucleic acid: Ob / ObR molecular hybrid. The presence of hybridized nucleic acids, if any, is then detected. Such detection schemes can be used to immobilize nucleic acids from the cell type or tissue of interest on a solid support, such as a membrane, or a plastic surface or polystyrene beads, such as in a microtiter plate. In this case, after incubation, the unannealed labeled nucleic acid reagent is easily removed. Remaining annealed sign Ob
Alternatively, detection of the ObR nucleic acid reagent can be performed using standard techniques well known to those of skill in the art. To determine if an Ob or ObR gene mutation is present,
The nucleic acid reagent annealed Ob or ObR gene sequences can be compared to the annealing pattern expected from the normal Ob or ObR gene sequences.

Alternative diagnostic methods for detecting Ob or ObR gene-specific nucleic acid molecules in patient samples or other suitable cell sources include, for example, PCR (experimental embodiment: Mul).
lis, KB, 1987, U.S. Pat. No. 4,683,202), followed by detection of the amplified molecule using techniques well known to those of skill in the art. To compare the resulting amplified sequence with the sequence expected if the nucleic acid to be amplified contains only normal copies of the Ob or ObR gene, to determine if the Ob or ObR gene mutation is present. You can

In addition, well-known genotyping techniques can be performed to identify individuals with Ob or ObR gene mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (FRLPs) with sequence variants at one of the recognition sites for the specific restriction enzyme used.

In addition, improved methods for analyzing DNA polymorphisms that can be used to detect Ob or ObR gene mutations have been described in the literature, and these methods have been shown to change between restriction enzyme sites. It takes advantage of the existence of a small number of short tandem repeat DNA sequences. For example, Weber (US Pat. No. 5,075,217, which is incorporated herein by reference in its entirety), described length polymorphisms in blocks of (dC-dA) n- (dG-dT) n short repeats. Based on DNA markers are listed. (dC-dA) n- (dG-
The average interval of dT) n blocks is estimated to be 30,000-60,000 bp. Such closely spaced markers show a high frequency of co-inheritance, eg Ob or ObR.
It is of significant utility in detecting genetic mutations, such as mutations within genes, and in diagnosing diseases and disorders associated with Ob or ObR mutations.

Also, Caskey et al. (US Pat. No. 5,364,759, which is incorporated herein by reference in its entirety), describe a DNA profiling assay for detecting short avian and tetra Ob or ObR genes. . The method involves extracting the DNA of interest, such as the Ob or ObR gene, amplifying the extracted DNA and labeling the repeat sequences to generate a genotype map of the individual's DNA.

Ob or ObR By detecting and measuring transcription respectively, Ob or ObR
Gene expression levels can also be assayed. For example, RNA from a cell type or tissue, such as brain cells known or suspected to express the Ob or ObR gene, in particular spinal cells, can be hybridized or hybridized as described above.
It can be isolated and tested using PCR technology. Isolated cells can be derived from cell culture or patients. Analysis of cells harvested from culture is a necessary step in the evaluation of cells to be used as part of cell-based gene therapy technology, or alternatively to test the effects of compounds on Ob or ObR gene expression. There are cases. Such analysis can reveal both quantitative and qualitative aspects of the expression pattern of the Ob or ObR genes, including activation or inactivation of Ob or ObR gene expression.

In one embodiment of such a detection scheme, cDNA is synthesized from the RNA of interest (eg, by reverse transcribing the RNA into cDNA). The sequence within the cDNA is then used as a template for nucleic acid amplification reactions such as PCR amplification reactions. Synthesis initiation reagents (eg, primers) in the reverse transcription and nucleic acid amplification steps of this method
The nucleic acid reagent used as is selected from the Ob or ObR nucleic acid reagents well known to those skilled in the art. The preferred length of such nucleic acid reagents is at least 9-30 nucleotides. Nucleic acid amplification can be performed with radioactively or non-radioactively labeled nucleotides in order to detect the amplified product. Alternatively, a sufficiently amplified product can be prepared so that it can be visualized by standard ethidium bromide staining or other suitable nucleic acid staining methods.

In addition, such Ob or ObR gene expression assays are performed “in situ”, ie, without the need to purify the nucleic acid, tissue sections (fixation and / or fixation) of patient tissue obtained from biopsy or excision. Or frozen). Nucleic acid reagents well known to those skilled in the art can be used as probes and / or primers for such in situ procedures (eg Nuovo, G.
J., 1992, "PCR In situ Hybridization: Protocols And Applications", Rave
n Press, NY).

Alternatively, if sufficient quantities of suitable cells can be obtained, standard Northern analysis can be performed to determine the mRNA expression levels of the Ob or ObR genes.

5.2.2 Detection of Ob or ObR Gene Products Antibodies directed against wild type or mutant Ob or ObR gene products, or conservative variants or peptide fragments thereof (these are described in Section 5.1.1 above). Can be used as a diagnostic and prognostic agent for bone disorders, as described herein. Using such a diagnostic method, Ob
Or abnormalities in ObR gene expression levels, or Ob and ObR structure and / or
Alternatively, abnormalities in the position of the temporal region, tissue, cell or hypothalamus can be detected.

For example, antibodies directed against the ObR ECD or Ob epitope can be used in situ to detect ObR or Ob expression patterns and levels in the body. Such antibodies can be labeled with, for example, radiopaque compounds or other suitable compounds and labeled with ObR or Ob expressed in the body using such methods as X-ray, CAT scan or MRI. Subjects can be injected to visualize binding. Labeled antibody fragments, such as Fab or single chain antibodies containing a minimal portion of the antigen binding region, may be used to facilitate crossing the blood-brain barrier for this purpose and to label ObR expressed in the brain. Is preferred to enable.

In addition, any Ob or ObR fusion protein or Ob or ObR conjugated protein whose presence can be detected can be detected and can be administered. For example, a labeled Ob or ObR fusion or conjugated protein with a radiopaque compound or other suitable compound can be administered and visualized in vivo as discussed above for labeled antibodies. .
Furthermore, such fusion proteins can be utilized in in vitro diagnostic procedures.

Alternatively, immunoassays or fusion protein detection assays as described above can be utilized in vitro on biopsy and autopsy samples to allow evaluation of Ob or ObR expression patterns. Such assays are Ob or ObR
It is not limited to the use of antibodies to define any individual epitope of The use of these labeled antibodies will yield useful information regarding translation of Ob or ObR and intracellular trafficking to the cell surface and can identify defects in processing.

The tissues or cell types to be analyzed are generally those known or expected to express the Ob or ObR genes, such as the hypothalamus and spinal plexus cells for ObR; And the fat cells for Ob. The protein isolation methods employed herein are described in, for example, Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory M.
anual ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)
As described in. This document is incorporated herein by reference in its entirety. Isolated cells can be derived from cell culture or patients. Analysis of cells harvested from culture is needed in the evaluation of cells that could be used as part of, or as an alternative to, cell-based gene therapy techniques to test the effects of compounds on the expression of the Ob or ObR genes. It may be a different stage.

An antibody or fragment of an antibody useful in the invention, eg, as described in Section 5.1.1 above, may be used to quantify the presence of the Ob or ObR gene product, or conservative variants or peptide fragments thereof. Alternatively, it can be detected qualitatively. This can be done, for example, by immunofluorescence assay techniques employing fluorescently labeled antibodies (see below in this section) with light microscopy, flow cytometry or fluorometric detection. Such techniques are particularly preferred when such Ob or ObR gene products are expressed on the cell surface.

Antibodies (or fragments thereof), or Ob or ObR fusion or conjugated proteins useful in the present invention may further comprise an Ob or ObR gene product, as in immunofluorescence assay, immunoelectron microscopy or non-immunoassay. , Or for in situ assays of conserved variants or peptide fragments thereof, or Ob
It can be histologically adopted for binding (in the case of labeled Ob fusion protein).

In situ detection can be performed by separating the histological examination material from the patient and applying it to the labeled antibody or fusion protein of the present invention. antibody(
Or fragments) or fusion proteins are preferably applied by overlaying a labeled antibody (or fragment) on a biological sample.
The use of such a procedure makes it possible to determine not only the presence or Ob binding of the Ob or ObR gene product, conservative variant or peptide fragment, but also its distribution in the examined tissue. Using the present invention, one of skill in the art can modify a wide range of histological methods to achieve such in situ detection.

Immunoassays and non-immunization assays for the Ob or ObR gene products, or conservative variants or peptide fragments thereof, typically involve biological fluids (
Samples such as serum or cerebrospinal fluid), tissue extracts, freshly harvested cells, or cell lysates incubated in cell culture can be treated with the Ob or ObR gene product, or conserved variants or peptide fragments thereof. Would be incubated in the presence of a identifiable detectably labeled antibody and the bound antibody detected by a number of techniques well known in the art.

The biological sample can be contacted with and immobilized on a solid support or carrier, such as nitrocellulose, or cells, cell particles or other solid support on which soluble proteins can be immobilized. The support can then be washed with a suitable buffer and subsequently treated with a detectably labeled Ob or ObR antibody or Ob or ObR fusion protein. The support can then be washed a second time with buffer to remove unbound antibody or fusion protein. The amount of label bound to the solid support can then be detected by conventional means.

By “solid support or carrier” is intended any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier may be soluble or insoluble to some extent for the purposes of the present invention. The support material can have any possible structural shape so long as the bound molecule can bind the antigen or antibody. Thus, the support may be spherical in shape, such as in the case of beads, or cylindrical, such as in the case of the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip or the like. Examples of preferable supports include polystyrene beads. One of ordinary skill in the art will know or will be able to ascertain other supports suitable for binding the antigen or antibody by using routine laboratory methods.

The binding activity of a given lot of Ob or ObR antibody or Ob or ObR fusion protein can be determined by a well-known method. One of ordinary skill in the art will be able to determine optimal assay conditions that can work for each determination by routine experimentation.

Regarding antibodies, one way in which the Ob or ObR antibody can be detectably bound is by linking it to an enzyme and using it in an enzyme immunoassay (EIA) (Voller, A., "
The Enzyme Kinked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizon
s 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville
Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31: 507-520; Butler, JE, 19
81, Meth. Enzymol. 73: 482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay.
, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (Eds.), 1981, Enzyme Imm
unoassay, Kgaku Shoin, Tokyo). The enzyme that binds to the antibody will react with a suitable substrate, preferably a chromogenic substrate, to produce a chemical moiety that can be detected, eg, by spectrophotometric, fluorometric or macroscopic means. Enzymes that can detectably label antibodies include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkali Phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose
-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase are included, but are not limited to. Detection can be carried out by a calorimetric method that employs a chromogenic substrate for the enzyme. Detection can also be performed by visual comparison of the extent of enzymatic reaction of the substrate with similarly prepared standards.

Detection can also be performed using various other immunoassays. It is also possible to detect by use of radioimmunoassay (RIA), for example by radiolabelling the antibody or antibody fragment (eg Weintraub, B., Principles of
Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniqu
es, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected by such means as using a gamma counter or scintillation counter, or by autoradiography.

The antibody can also be labeled with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate,
Rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

Antibodies can also be detectably labeled using fluorescent emitting metals such as ¹⁵² Eu or other lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of chemiluminescent attached antibody is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeled compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which catalytic proteins increase the efficiency of chemiluminescent reactions. The presence of bioluminescent proteins is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

5.3 Screening for compounds useful in the treatment, diagnosis and prevention of bone disorders
Thesssays The invention also provides screening methods (eg, assays) for identifying compounds that affect bone disease. The invention further provides leptin receptors, including small molecules, large molecules and antibodies, and nucleotide sequences (eg, antisense and ribozyme molecules) that can be used to inhibit leptin and leptin receptor gene expression, and leptin and leptin receptors. Leptin and leptin receptor agonists and antagonists, including genes or regulatory sequence replacement constructs designed to enhance gene expression (eg, expression constructs that place the leptin and leptin receptor genes under the control of a strong promoter system) Includes. Such compounds can be used to treat bone disorders.

In particular, cellular and non-cellular compounds that can be used to identify compounds that interact with leptin and the leptin receptor, ie compounds that modulate the activity of leptin and the leptin receptor and / or that bind to the leptin receptor. A sex assay has been described. Using cell-based assays, compounds or compositions that affect the signal transduction activity of leptin and the leptin receptor are tested against intracellular factors that either bind to the leptin receptor or are associated with the leptin signal transduction pathway. Can be identified. Such cell-based assays of the invention of the invention utilize cells, cell lines, or engineered cells or cell lines that express leptin or the leptin receptor. The cells can be further engineered to introduce receptor molecules linked to the signal by activated leptin receptors to assist in identifying leptin and compounds that modulate leptin receptor signaling activity.

The invention also includes the use of cell-based or cell lysate assays (eg, in vitro transcription or translation assays) to screen for compounds or compositions that modulate leptin and leptin receptor gene expression. To do. To this end, a construct containing a reporter sequence linked to a leptin or leptin receptor gene modulation element is used in engineered cells or cell lysates to modulate the expression of the reporter gene product at the transcriptional level. You can screen about. For example, such assays are used to identify compounds that modulate expression or activity of leptin and transcription factors associated with leptin receptor gene expression, or 3
The activity of the heavy helix polynucleotide could be tested. Alternatively, using engineered cells or transcript extracts, leptin and leptin receptor
Compounds (including antisense and ribozyme constructs) that modulate translation of mRNA transcripts and thus affect expression of the leptin receptor can be screened.

The assays below describe compounds that interact with (eg, bind to) Ob or ObR, including but not limited to ECD or CD of ObR; Ob or ObR (O
Compounds that interact with (eg, bind to) intracellular proteins that interact with (including but are not limited to) bR TM and CD; Ob or ObR and ObR
Designed to identify compounds that interfere with transmembrane or intracellular protein interactions associated with mediated signal transduction; and compounds that modulate the activity of Ob or ObR gene expression or modulate the level of Ob or ObR To be done. In addition, assays are available that identify compounds that bind to the Ob or ObR gene regulatory sequences (eg, promoter sequences), and compounds that can modulate Ob or ObR gene expression. For example Platt, K. A
., 1994, J. Biol. Chem. 269: 28558-28562. Upon identification, the compounds can be tested for their ability to modulate leptin signaling in vivo or in vitro, and also the ability to modulate bone mass (ie increase or decrease bone mass) and the corresponding non-disease. The ability to treat bone disorders characterized by increased or decreased bone mass relative to bone can be tested.

Therefore, according to this aspect of the invention, modulation of bone mass in a mammal (
Increasing or decreasing) ability to identify a compound to be tested, which method comprises: (a) contacting the test compound with a polypeptide; and (b) whether the test compound binds the polypeptide. And thus identifying a compound to be tested for its ability to modulate bone mass if the test compound binds the polypeptide, wherein the polypeptide is a leptin polypeptide and a leptin. Selected from receptor polypeptides.

Alternatively, a method of identifying a compound that modulates (increases or decreases) bone mass in a mammal, the method comprising: (a) contacting a test compound with a polypeptide; (b) binding the polypeptide. A test compound is identified; and (c) the test compound in (b) is administered to a non-human mammal and the test compound is administered to the corresponding bone mass of an untreated non-human control mammal. Determining whether it modulates bone mass in, wherein the polypeptide is selected from a leptin polypeptide and a leptin receptor polypeptide such that the test compound binds the polypeptide. , Identify compounds that modulate bone mass.

According to this and another aspect of the invention, a non-human control mammal, as used herein, is intended to mean the corresponding mammal to which the test compound was not administered. There is.

According to yet another aspect of the invention is a method of identifying a compound to be tested for its ability to modulate (increase or decrease) bone mass in a mammal, which method comprises: (a) the test compound being a leptin Contacting the polypeptide and the leptin receptor polypeptide for a period of time sufficient to form a leptin / leptin receptor complex; and (b) measuring leptin / leptin receptor complex levels and, as a result, determined If the levels differ from those measured in the absence of the test compound, then identifying the compound to be tested for its ability to modulate bone mass.

According to such and other aspects of the invention, the production of the leptin / leptin receptor complex can be performed, for example, by isolating the complex and performing various assays well known to those of skill in the art (eg, Western blot). Can be measured by measuring the production amount of the complex.

According to another aspect of the present invention, there is provided a method for confirming whether a test compound has the ability to reduce bone mass in a mammal, which comprises (a) expressing the test compound by expressing a functional leptin receptor. And (b) determining whether the test compound activates the leptin receptor, and the test compound activates the leptin receptor, the test compound activates the bone mass in a mammal. A method is provided that is confirmed to have the ability to reduce

According to such an aspect and another aspect of the invention, the functional leptin receptor is
It is a leptin receptor having a signal transduction ability after a ligand binds to the active site of the receptor. As used herein, activation of the leptin receptor means increased activity (ie, signal transduction) of the leptin receptor.

According to another aspect of the present invention, there is provided a method of identifying a compound that reduces bone mass in a mammal, comprising: (a) contacting a test compound with a cell expressing a functional leptin receptor, It is determined whether the test compound activates the leptin receptor, (b) the test compound confirmed to activate the leptin receptor in (a) is administered to a non-human animal, and the test compound is It is determined whether the animal's bone mass is reduced compared to the corresponding bone mass of a control non-human animal, so that if the test compound reduces bone mass, the bone mass is reduced in the mammal. Methods are provided in which the compounds that are to be identified are those that are identified.

According to another aspect of the present invention, there is provided a method of determining whether a test compound has the ability to increase bone mass in a mammal, comprising: (a) adding a leptin polypeptide and a test compound to a functional leptin. Contacting cells expressing the receptor, and (b) determining whether the test compound reduces activation of the leptin receptor relative to that observed in the absence of the test compound, and leptin Methods are provided in which a test compound that decreases receptor activation is identified as a test compound having the ability to increase bone mass in a mammal.

According to yet another aspect of the invention, a method of identifying a compound that increases bone mass in a mammal comprising: (a) expressing a leptin polypeptide and a test compound at a functional leptin receptor. It was measured whether or not the test compound reduces the activation of the leptin receptor by contacting with cells, and the test compound confirmed to reduce the activation of the leptin receptor in (b) and (a) was used as a non-human animal. To determine whether the test compound increases bone mass of the animal as compared to the corresponding bone mass of a control non-human animal, such that the test compound increases bone mass. In some cases, methods are provided wherein compounds that increase bone mass in a mammal are identified.

According to another aspect of the present invention, activation of the leptin receptor is also linked to phosphorylation Stat.
There is a method of determining by measuring the level of 3 polypeptides. Stat3 polypeptide, an effector downstream of leptin signaling in target cells (Tartaglia et al., 1995, Cell 83, 1263-1271; Baumann et al., 1996, Proc. Na
tl. Acad. Sci. USA 93, 8374-8378; Ghilardi et al., 1996, Proc. Natl. Acad. Sc
i. USA 93. 6231-6235; Vaisse et al., 1996, Nat Genet 14, 95-97) is phosphorylated after activation of the leptin receptor by leptin.

Compounds that can be screened according to the present invention include, but are not limited to, those that bind to Ob or ObR and mimic the activity elicited by a natural ligand (ie, agonist), or Peptides, antibodies and fragments thereof, and other organic compounds (eg, peptidomimetics) that inhibit the activity elicited by a ligand (ie, an antagonist); and bind to and “neutralize” a natural ligand. , Peptides, antibodies or fragments thereof, and other organic compounds that mimic the ECD (or part thereof) of ObR. Additional compounds that can be screened in accordance with the present invention include, but are not limited to, compounds that interact with Ob and inhibit the transport of Ob across the blood brain barrier. This compound prevents Ob from activating ObR.

Such compounds include, but are not limited to, peptides such as soluble peptides (including, but not limited to, members of a random peptide library; eg, Lam, KS et al. 1991, Nature 354: 82-84; Houg
hten, R. et al., 1991, Nature 354: 84-86), and combinatorial chemistry-derived molecular libraries made from D- and / or L-configured amino acids, phosphopeptides (but not limited to). No, but includes members of a random or partially degenerate directed phosphopeptide library; eg S
ongyang, Z. et al., 1993, Cell 72: 767-778), antibodies (including but not limited to polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, anti-ididotypic antibodies). , Chimeric or single chain antibodies, and FAb, F (ab ′) ₂ , and FAb expression library fragments, and epitope binding fragments thereof), and small organic or inorganic molecules.

Other compounds that can be screened according to the present invention include, but are not limited to, crossing the blood-brain barrier into suitable cells (eg, choroid plexus or cells of the hypothalamus), Ob or O involved in ObR signaling pathway
small organic molecules capable of affecting the expression of the bR gene, or some other gene (eg, by interacting with regulatory regions or transcription factors involved in gene expression); or affecting the activity of ObR ( Such compounds include, for example, acting by inhibiting or enhancing the enzymatic activity of CD) or the activity of some other intracellular factors involved in the ObR signaling pathway, such as gp130.

Computer modeling and search techniques allow for the identification of compounds that can modulate the expression or activity of Ob or ObR, or the modification of previously identified compounds. By identifying such compounds or compositions, the active site or region is identified. Such active sites are typically ligand binding sites, eg, the interaction domain of Ob with ObR itself.
Active sites can be identified using methods well known in the art, such as, for example, from the amino acid sequence of peptides, from the nucleotide sequence of nucleic acids, or related compounds or compositions and their natural Methods from studies of complexes with the ligands of. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where the ligand complex on the factor is found. Next, the three-dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine the complete molecular structure. On the other hand, solid phase or liquid phase NMR can be used to measure specific intermolecular distances. Any other experimental method for structure determination can be used to obtain partial or complete geometric structures. This geometry may be measured together with the ligand complex, and natural or artificial ligand complexes may increase the accuracy of the active site structure being determined.

When structures are determined with incomplete or inadequate accuracy, computer-based numerical modeling methods can be used to complete the structure or improve accuracy. Any recognition modeling method may be used, including such parameterized models specific to particular biopolymers such as proteins or nucleic acids,
Molecular dynamics model based on molecular behavior calculation, thermal ensemble
ble) based statistical mechanics model, or composite model. Many types of models require standard intermolecular force fields, which represent the forces between the constituent atoms and the groups, and can be chosen from the force fields known in physical chemistry. This incomplete or inaccurate experimental structure may serve as a constraint on the complete or more accurate structure calculated by the modeling method described above.

Finally, by determining the structure of the active site, either experimentally by modeling or by combination, candidate modulating compounds are searched for databases containing compounds that match the information regarding molecular structure. Can be identified by By such a search, a compound having a structure that matches the determined structure of the active site and has a structure that interacts with the group defining the active site is searched for. Although such a search may be performed by one's own hand, it is preferable to use a computer. The compounds found by this search are potential Ob or ObR modulating compounds.

Alternatively, these methods can be used to identify improved regulatory compounds from previously known regulatory compounds or ligands. The composition of known compounds can be modified and the structural effects of the modifications can be determined using the experimental and computer modeling methods described above applied to the novel compositions. The modified structure is then compared to the active site structure of the compound to determine if the fit or interaction was improved. In this way, global changes in the composition, for example due to changes in side groups, can be readily evaluated to provide modified modulator compounds or ligands with improved specificity or activity.

Additional experimental and computer modeling methods useful in identifying regulatory compounds based on the identification of Ob, ObR active sites and associated transducing and transcription factors will be apparent to those of skill in the art.

As an example of a molecular modeling system, the CHARMm and QUANTA programs (Polygen
Corporation, Waltham, Mass.). CHARMm performs energy minimization and molecular dynamics functions. QUANTA performs molecular structure construction, graphic modeling, and analysis. QUANTA enables interactive construction, modification, visualization and analysis of the mutual behavior of molecules.

Numerous articles outline computers that model drugs that can interact with specific proteins. For example, Rotivinen et al., 1988, Acta Pharmaceutic.
al Fennica 97: 159-166, Ripka, New Scientist 54-57 (Jun. 16,1988); McKin
aly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29: 111-122; Perr
y 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. Lond. 236: 125-140 and 141-162; and regarding model receptors for nucleic acid components, Askew et al., 1989, J. Am. Chem. Soc. 111: 1082-1090. Other computer programs that screen and visually display chemicals are BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontari
, Canada), and Hypercube, Inc. (Cambridge, Ontario). They are primarily designed for application to drugs specific to particular proteins, but can also be applied to the design of drugs specific to regions of DNA or RNA (if such regions are identified).

Although described above with respect to the design and production of compounds capable of altering binding, known compounds, such as natural or synthetic chemicals, and live biologically active materials (eg, proteins) can be used. The rally can also be screened for compounds that are inhibitors or activators.

The compounds identified by the assays as described herein may be useful, for example, in creating the biological function of the Ob or ObR gene products, and for ameliorating bone disease. For example, an assay for testing the efficacy of compounds identified by the methods as described in Sections 5.3.1 to 5.3.3 is described below in Section 5.3.4.

5.3.1. In vitro screening assay for compounds that bind to Ob and ObR
The essay in vitro system may be designed to identify compounds capable of interacting with (eg, binding to) Ob and ObR, including but not limited to ECD or ObR CD. The identified compound may be, for example, wild type and / or mutated.
It may be effective in modulating the activity of the Ob or ObR gene product and in refining the biological function of Ob or ObR. It could also be used in a screen to identify compounds that disrupt normal Ob and ObR interactions. Alternatively, it may disrupt such interactions within the identified compound itself.

The principles of the assay used to identify compounds that bind to Ob or ObR include providing a reaction mixture of Ob or ObR and its test compound. Sufficient conditions and time should be provided that the two components interact, bind, and form a complex that can be removed and / or detected from the reaction mixture. The Ob or ObR species used will depend on the goals of the screening assay. For example, if one is looking for an agonist of the natural ligand, a standard length ObR, or a soluble truncated ObR, such as where TM and / or CD is deleted from the molecule, or a peptide corresponding to the ECD, or Fusion proteins containing the ObR ECD fused to a protein or polypeptide that provide advantageous factors in the assay system (eg, labeling, sequestration of the resulting complex, etc.) are available. If it is desired to identify compounds that interact with the ObR cytoplasmic region, peptides corresponding to ObR CD and fusion proteins containing ObR CD can be used. In addition, Ob or a soluble form of Ob can be used when looking for compounds that prevent Ob from entering the blood-brain barrier.

The screening assay can be performed in a variety of ways. For example, in one method of performing such an assay, an Ob or ObR protein, polypeptide, peptide, or fusion protein or test substance thereof was immobilized on a solid phase and at the end of the reaction was immobilized on the solid phase. , Ob or ObR and its test compound complex are detected. In one embodiment of such a method, the Ob or ObR reactant may be immobilized on a solid surface and the test compound, although not immobilized, may be directly or indirectly labeled.

As a practical matter, microtiter plates can conveniently be utilized as a solid phase. The components to be immobilized can be non-covalently or covalently immobilized. Non-covalent attachment may occur simply by coating the solid surface with a solution of the protein and then drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, that has a specific effect on the immobilization of the protein can be used to immobilize the protein on a solid surface. The surface may be prepared and stored in advance.

To perform the assay, the non-immobilized component is added to the coated surface containing the immobilized component. After the reaction is complete, the unreacted components are removed (eg by washing), but under conditions such that any complex formed remains immobilized on its solid surface. The detection of complexes immobilized on the solid surface can be done by several methods. Where surface-immobilized label was detected, where pre-immobilized components were pre-labeled, it indicates that a complex had formed. Indirect labeling can be used to detect surface-immobilized complexes, where pre-immobilized components have not been previously labeled. For example, a labeled antibody having a specific effect on a component which is not immobilized in advance is used. (The antibody may then itself be labeled directly or indirectly with a labeled anti-Ig antibody.) Alternatively, the reaction is carried out in the liquid phase. , The reaction product is separated from non-reacted components,
The complex can be detected. For example, a specific immobilized antibody that immobilizes any complex of the Ob or ObR protein, polypeptide, peptide or fusion protein or test compound thereof formed in solution, and other of the complexes that can be produced. A labeled antibody, which has a specific effect on detecting a complex in which the components of the above are immobilized, is used.

Alternatively, cell-based assays can be used to identify compounds that interact with Ob or ObR. To this end, cell lines expressing Ob or ObR or cell lines (eg COS cells, CHO cells, fibres) which have been genetically engineered to express Ob or ObR (eg by transfection or transduction of Ob or ObR DNA). Blast cells, etc.) can be used. The interaction of the test compound with the ECD of, for example, the ObR expressed by the host cell can be determined by comparison or competition with native Ob.

5.3.2 Assays for Proteins That Interact with Ob and ObR Any method suitable for detecting protein-protein interactions will identify transmembrane or intracellular proteins that interact with Ob or ObR. May be used for Some traditional methods of potential use were obtained from cell lysates and Ob or ObR to identify proteins in lysates that interact with immunoprecipitation, cross-linking, and Ob or ObR. , Cell lysates or protein gradients or chromatographic columns for mutual purification. In these assays, the Ob or ObR components used are of standard length, soluble derivatives lacking a membrane anchoring region (eg, truncated ObR, where the TM is deleted, resulting in CD
Becomes a truncated molecule containing ECD fused to), a peptide corresponding to that CD,
It can be a fusion protein containing a CD of Ob or ObR. Once sequestered, such intracellular proteins can be identified and then used in conjunction with standard techniques to identify the proteins with which they interact. For example, Ob
Or at least a part of the amino acid sequence of the intracellular protein that interacts with ObR, using a technique well known to those skilled in the art, such as using Edman degradation technology,
Can be confirmed (Reference example: Creighton, 1983, “Proteins: Structures an
d Molecular Principles ”, WH Freeman & Co., NY, pp.34-49). The resulting amino acid sequence can be used as an indicator for the production of a mixture of oligonucleotides. It can be used to screen the encoding gene sequences, which may be accomplished, for example, by standard hybridization or PCR techniques. Oligonucleotide mixture generation and screening techniques are well known (see Example: Ausube
l, supra., and PCR Protocols: A Guide to Methods and Applications, 1990,
Innis, M. et al., Eds. Academic Presss, Inc., New York).

In addition, methods such as the simultaneous identification of multiple genes encoding transmembrane or intracellular proteins that interact with Ob or ObR may be used. These methods include, for example, a method of probing the expression library by a method similar to the known antibody probe technique for the λgt11 library. For this purpose, a labeled Ob or ObR protein, or an Ob or ObR polypeptide, peptide or fusion protein, such as an Ob or ObR polypeptide or a label (eg enzyme, fluorine, photoprotein, or dye) fused to Ob or
It is the ObR region or the Ig-Fc region.

One of the methods for detecting protein interactions in vivo, the two-hybrid system, has been described in detail, but is given by way of illustration and not limitation. One explanation of this system has already been detailed (Ch
ien et al., 1991, Proc. Natl. Acad. Sci. USA, 88-9578-9582), and Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, a plasmid encoding two interacting (two-hybrid) proteins is constructed. One of them, the plasmid, consists of nucleotides encoding the DNA binding region of the transcriptional activator protein fused to the Ob or ObR nucleotide sequence, encoding the Ob or ObR, Ob or ObR polypeptide, peptide or fusion protein. The other plasmid consists of nucleotides encoding the active region of the transcriptional activator protein fused to complementary DNA encoding an unknown protein that has been recombined into this plasmid as part of a complementary DNA library. The DNA binding region fusion plasmid and the complementary DNA library are transformed into a strain of yeast (Saccharoyces cerevisiae). The strain contains a reporter gene (eg HBS or lacZ),
The regulatory region of the gene contains the binding site for the transcriptional activator. Neither hybrid protein alone can activate the transcription of the reporter gene. DN
The A-binding domain hybrid cannot be activated because it does not provide the activation function, and the activation-region hybrid cannot be activated because it cannot set at the binding site of the activator. This two-hybrid protein interaction reconstitutes the functional activator protein, resulting in expression of the reporter gene. This is detected in the reporter gene product assay.

The two-hybrid system or related methodology described above may be used to screen activation region libraries to screen for proteins that interact with the "bait" gene product. By way of illustration and not limitation, Ob or ObR may be used as a bait gene product. The entire genomic or complementary DNA sequence is fused to the DNA encoding the activation region. A plasmid encoding a hybrid of this library and the bait Ob or ObR gene product fused to the DNA binding region is co-transformed into a yeast reporter strain. The resulting transformants are screened for transformants expressing the reporter gene. For example, but not by way of limitation
Ob or ObR (or a region of ObR) open reading frame,
The bait Ob or ObR gene sequence encodes the DNA-binding region of GAL4 protein D
It can be cloned into a vector for translational fusion to NA. These colonies are isolated and the library plasmid responsible for reporter gene expression is isolated. DNA sequencing is then used to identify the protein encoded by the library plasmid.

Cell-based complementary DNA libraries, in which proteins that interact with the bait Ob or ObR gene products should be detected, can be made using methods routinely practiced in the art. According to the identification system described here, for example,
Complementary DNA fragments, such that they fuse to the transcriptional activation region of GAL4 during translation,
Can be inserted into the vector. This library is a decoy Ob
Alternatively, together with the fusion plasmid of the ObR gene and GAL4, a yeast strain containing the lacZ gene driven by a promoter containing the GAL4 activating sequence is co-transformed. A complementary DNA-encoded protein fused to the GAL4 transcriptional activation region and interacting with the bait Ob or ObR gene product reconstitutes the active GAL4 protein, thereby driving the expression of the HIS3 gene. Colonies expressing HIS3 can be detected by growing on petri dishes containing histidine-deficient, semi-solid, agar-based medium. The complementary DNA can then be isolated from these strains and used to generate and sequester the bait Ob or ObR gene interacting protein using methods routinely practiced in the art. is there.

5.3.3 Compounds that interfere with Ob and ObR / intracellular or ObR / transmembrane macromolecular interactions
Macromolecules that interact with the assay Ob or ObR of a product are referred to herein as “binding partners”. These binding partners are thought to be involved in the ObR signaling pathway. Therefore, there is a role of Ob or ObR in the regulation of bone disease. As a result, O
It is desirable to use b to identify compounds that interfere with or separate the interaction of such binding partners. This may be useful in regulating ObR activity,
Inhibits skeletal disorders associated with activity.

The basic rules of the assay procedure have always been to identify compounds that interfere with the interaction with Ob or ObR. And their binding partners or partners include providing a reaction mixture comprising Ob or ObR proteins, polypeptides, peptides, or melting proteins, as described above. And when conditions and time are sufficient, the binding partners allow both to interact and bind, thus forming a complex. To analyze the compounds for inhibitory activity, the reaction compounds are adjusted in the presence or absence of the assay compound. The analyte compound may initially be included in the reaction compound. Or Ob, or a portion of ObR and its addition of binding partners may be added at once. Control reaction compounds are incubated without assay compound or with placebo. The formation of any complex and binding partner with the Ob or ObR moiety is then detected. In the control reaction, the formation of the complex, but the inclusion of the analytical compound, but not the reactive compound, indicates that the compound interferes with Ob or ObR, and the binding partner of the interaction. Moreover, the formation of the complex within the reaction compound containing the analyte and the standard Ob or ObR protein results in
It may be compared to complex formation within a reactive compound containing ObR. Although this comparison is preferred for identifying compounds that dissociate the mutant interaction, Ob
Or, it is not preferable for ObR, and such a case becomes important.

Assays for compounds that interfere with Ob or ObR interactions and binding partners are processed in heterogeneous or homogeneous formats. For heterogeneous assays, use the solid state
Immobilizing either the Ob or ObR partial product, or the binding partner, and the detection of complexes that become immobilized at the end of the reaction are included. For analysis of the same structure, all reactions are continued in liquid form. In either method, the order of addition of reactants can be changed to obtain various information about the compound being analyzed. For example, analyte compounds that interfere with competitive interactions can be identified by treating the reaction in front of the analyte. That is, the analyte was added to the reaction mixture in advance, or Ob or ObR
Partial and interactive binding partners, with simultaneous addition. Alternatively, an analytical compound that separates the preformed complex, for example, a compound with a high binding constant that displaces one of the components from the complex, adds the analytical compound to the reaction mixture after the complex is formed. It can be analyzed. Various configurations will be briefly described below.

In a heterogeneous assay procedure, either Ob or a portion of ObR, or the binding partner of the interaction, is immobilized on the surface of the solid. On the other hand, the non-fixed molecule is directly or indirectly labeled. As a practical matter, microtiter plates are conveniently used. The immobilized molecule can be non-covalent or immobilized by covalent attachment. Non-covalent conjugates can be easily accomplished by coating the surface of the solid with the Ob or ObR gene product or binding partner and drying. Alternatively, the properties of immobilizing antibodies with respect to the molecule to be immobilized can be used to immobilize the molecule on the surface of a solid. The surface can be prepared and stored in advance.

To perform the assay, the immobilized molecular partner is exposed to cover the surface, with or without analytical components. After the reaction is complete, the unreacted components are removed (eg, washed), and any complex formed remains immobilized on the surface of the solid. Detection of complexes anchored on the surface of solids can be accomplished in a number of ways. When the non-immobilized molecule was pre-labeled, detection of the label immobilized on the surface indicates that the complex had formed.
If the non-immobilized molecule is not previously labeled, indirect labeling is used to detect the complex immobilized on the surface. For example, a labeled antibody molecule is used, which refers to the molecule that is not immobilized initially. (The antibody is sequentially labeled directly or indirectly with a labeled anti-Ig antibody), depending on the order of addition of the reaction components, an analytical compound that inhibits complex formation, or An analyte compound is detected that separates the complex formed in.

Alternatively, the reaction is a reaction product separated from unreacted components of the analytical compound,
And with or without detected complexes can be processed in the liquid state. For example, an immobilized antibody molecule is used as one of the binding components to immobilize any complex formed in solution. And labeled antibody molecules with respect to other partners are used to detect the immobilized complex. Again, depending on the additional order of reaction to the liquid state, analyte compounds are identified that inhibit the complex or separate the preformed complex.

In an alternative embodiment of the invention, a homogeneous assay can be used. in this way,
A preformed complex of a portion of Ob or ObR and the binding partner of the interaction is prepared for labeling either Ob or ObR or its binding partner. However, the signal generated by labeling is complex formation (
For example, Rubenstein uses this method in an immunoassay, US Pat. No. 4,109,49
(See No. 6). Addition of the analyte will generate one signal in the background when competing and displacing one of the molecules of the preformed complex.
In this way, analytes that segregate Ob or ObR / intracellular binding partner interactions can be identified.

[0171] In certain embodiments, the Ob or ObR fusion is immobilized. For example, Ob or ObR or peptide fragment, eg, corresponding to ObR CD, pGEX-5X
-1 etc., using a fusion vector, glutathione S transferase (
GST) gene. In this way, its binding activity is regulated by the resulting fusion protein. Once the interactive binding partner is removed, it is used to recover the monoclonal antibody using methods commonly practiced in the art. This antibody can be labeled with radioisotope 1. For example, by methods well practiced in the art. In a heterogeneous assay, for example, the GST-ObR fusion protein can be immobilized on glutathione agarose beads. The binding partner of the interaction is then in a way that allows the interaction or binding to occur,
It can be added with or without the analytical complex. At the end of the reaction time, unbound material is washed off and labeled monoclonal antibody is added to the method, allowing the conjugated components to bind. The Ob or ObR gene product, and the binding partner of the interaction, can be detected by measuring the amount of radioactivity remaining associated with glutathione agarose beads. Successful inhibition of the interaction by the analytical compound will reduce the measured radioactivity.

Alternatively, the GST-Ob / ObR melting protein and the binding partner of the interaction are:
They can be mixed together in a liquid lacking pure glutathione agarose beads. Analyte compounds can be added either while the molecules are allowed to interact, or afterwards. This compound is then added to glutathione agarose beads to wash away unbound material. Again, Ob or Ob
The extent of inhibition of the R / binding partner interaction can be detected by the addition of labeled liquid and measurement of the radioactivity binding to the beads.

In another embodiment of the invention, such a similar technique may be applied to Ob or ObR and / or interaction or binding partner (if the binding partner is a protein), which is one or two full-length proteins. Instead of one, the use of peptide fragments corresponding to the binding range is given. Any number of methods routinely practiced in the art can be used to identify and separate binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and protecting the disruption of binding in a co-immunoprecipitation assay. Compensating for mutations in the gene encoding the second molecule in the complex is then selected. Sequential analysis of the genes encoding each protein reveals mutations, which correspond to the range of proteins involved in the interactive binding. Alternatively, one protein can be immobilized on a solid surface using the methods described above, allowing it to interact and bind with a labeled binding partner. It has been treated with proteolytic enzymes such as trypsin. After washing, the purely labeled peptides that make up the binding domain can remain bound to pure substances that can be separated and identified by their amino acid sequences. Similarly, once the gene encoding the binding partner in the cell has been obtained, small gene splits can be processed to represent peptide fragments of the protein. This can then be analyzed for binding activity, and clearance, or synthesis.

For example, but not as a means of restriction, the Ob or ObR gene product can be immobilized on a solid material as described above to produce the GST-Ob or ObR protein, which can be glutathione agarose beads. Because it is possible to bind to. The binding partner of this interaction can be labeled with a radioisotope such as 35S and bound to a proteolytic enzyme such as trypsin. The fission products are then capable of binding when added to the immobilized GST-Ob or ObR melting protein. After washing the unbound peptide, the intracellular binding partners, labeled binding substances representing the binding domains, are eluted, clarified and analyzed for amino acid sequences by well known methods. The peptides thus identified can be produced or fused synthetically to the appropriate protein using recombinant DNA technology.

5.3.4 Assays for Identification of Compounds that Restore Bone Diseases Compounds include, but are not limited to, compounds identified by assay techniques, as described above in Sections 5.3.1 to 5.3.3. Those that are not, can be tested for their ability to treat bone disease and ameliorate symptoms of bone disease. The above assay
Compounds that bind Ob or ObR behavior (eg, leptin receptor agonists or antagonists) and ObR natural ligands, and compounds that abrogate ligand action or affect Ob or ObR gene behavior (including microparticles) By affecting Ob or ObR gene expression, eg, full-length expression of Ob or ObR, when a protein or small amount of organic microparticles affects or interferes with event conjugation,
Or the chamfered morphology is adjusted), the assay described
Modulates bR signaling (eg, compounds that influence signaling events along the stream, such as inhibitors or enhancers of tyrosine kinases, or Ob binding to ObR, which contributes to the transduction of signals activated by hostaphase action) It should be noted that it is also possible to identify compounds that do.
As an alternative, the above assay may also identify compounds that modulate Ob entry through the blood-brain barrier. The identification and use of such compounds affects other stages of the Ob or ObR signal transduction pathway. This includes the Ob or ObR genes and / or gene products. Also, by affecting this same pathway, Ob or Ob in the development of bone disorders
The effect of R can be adjusted. This is within the scope of the invention. Such compounds can be used as part of therapeutic methods for treating bone disorders.

Cellular reference systems are used to identify compounds that reverse bone disease. Such cell lines can include recombinant or non-recombinant cells, such as, for example, the Ob or ObR gene, eg, a cell line expressing the NTH3TT3L cell line. Furthermore, for ObR, for example, choroidal reticuloblasts, hypothalamic cells, or cell lines derived from choroidal reticulum or hypothalamus are used. Furthermore, expressing recipient cells (eg, COS cells, CHO cells, fibroblasts) are produced by genes and express functional OBs or OBRs and reactions of activation by natural OB ligands, eg chemical or expression. Type changes, induction of other recipient cell genes, alteration of ion influx (eg, Ca ++), tyrosine phosphorylation of recipient cell proteins, etc. are processed. These are used as the endpoint of the assay.

[0177] When utilizing such cell lines, cells can be exposed to, and sufficient time to expose, compounds that are believed to be fully condensed and exhibit the ability to restore bone damage. Induces recovery of bone damage in the treated cells. After exposure, the cells may be Ob or Ob
The expression of the R gene can be assayed to regulate degeneration. For example, Ob or ObR mRNA transcript or Ob or O expressed intracellularly.
By assaying cell isolates of bR protein (eg, Northern analysis). Thus, regulation of the Ob or ObR genes, or regulatory expression, is a good candidate for treatment. As an alternative, the cells may recover one or more bone disorders, such as a cellular phenotype, to become more normal, or even wild type,
It appears to be prone to developing symptoms of boneless dysfunction phenotype or excessive dysfunction. Furthermore, the expression and / or activity of components of the signal transduction pathway, the activity of this ObR part or the ObR signal transduction pathway itself can be assayed.

For example, after exposure, cell lysates can be assayed for the presence of tyrosine phosphorylation of host cell proteins and compared to lysates derived from unexposed control cells. The ability of test compounds to inhibit host cell protein tyrosine phosphorylation in these assay systems indicates that the test compounds inhibit signal transduction initiated by ObR activation. Cell lysates can be easily assayed using Western blotting, ie host cell proteins are lysed by gel electrophoresis, transferred and anti-phosphorylated detection antibodies (eg radiolabeled, fluorspar, enzymes, etc.). , An anti-phosphorylated antibody labeled with a signal-generating compound). (See also: Glenney et al., 1
988, J. Immunol. Methods 109: 277-285; Frackelton et al., 1983, Mol. Cell. Bio.
l. 3: 1343-1352). Alternatively, an ELISA format can be used where a particular host cell protein involved in the ObR signaling pathway is immobilized using an immobilized antibody specific for the target host cell protein and the presence of phosphorylation on the immobilized host cell protein. Alternatively, the absence is detected using labeled anti-phosphorylated antibodies (see King et al., 1993, Life Science 53: 1465-1472). In yet another approach, ion influx, such as calcium ion influx, can be measured as the endpoint of ObR-induced signaling.

[0179] In addition, animal-based bone injury systems have been described, for example, in ob, db, and Ob.
/ Db mice and can be used to identify compounds that can ameliorate conditions such as bone disorders. Such animal models can be used as test substrates for drugs, pharmaceuticals, treatments and interventions that can be effective in treating such disorders. For example, animal models have shown that a compound suspected of exhibiting the ability to ameliorate a bone disorder condition in sufficient concentration and for sufficient time to induce such treatment of the bone disorder condition in an exposed animal. Can be exposed. Animal response to irradiation can be monitored by assessing reversal of disorders associated with bone disorders such as osteoporosis. With respect to intervention, any treatment that reverses any aspect of symptoms such as bone disorders is considered a candidate for human bone disorder treatment intervention. The dose of test drug can be determined by determining a dose-response curve.

5.4 Compounds that Modulate Ob or ObR Expression or Activity Compounds that interact with (eg, bind to) Ob or ObR (eg, but not limited to ECD or CD of ObR), interact with Ob or ObR Compounds that interact with (e.g., bind to) intracellular proteins (e.g., but not limited to ObR TM and
CD), compounds that interfere with the interaction of Ob or ObR with transmembrane or intracellular proteins involved in ObR-mediated signaling, and modulate the activity of Ob or ObR gene expression, or of Ob or ObR Compounds that modulate levels have the ability to modulate levels of bone mass. More specifically, it reduces the level of Ob or ObR, inhibits the transport of Ob across the blood-brain barrier,
Alternatively, compounds that inhibit the binding of Ob to ObR will cause an increase in bone mass.

Examples of such compounds are leptin and leptin receptor agonists and antagonists. As used herein, a leptin receptor antagonist refers to a factor that neutralizes or interferes with, or otherwise reduces, the action or effect of a leptin receptor. Such antagonists may include compounds that bind to leptin or compounds that bind to leptin receptors. Such antagonists include leptin receptor output, an intracellular step in the leptin signaling pathway following binding of leptin to the leptin receptor, a downstream event that affects leptin / leptin receptor signaling. /
Compounds that neutralize, interfere with or reduce) (which do not occur at the level of ligand interaction). Leptin receptor antagonists include, but are not limited to, proteins, antibodies, small organic molecules or carbohydrates such as acetylphenol compounds, antibodies that specifically bind to leptin, antibodies that specifically bind to leptin receptor, And a compound comprising a soluble leptin receptor polypeptide sequence.

For example, leptin antagonists are agents and drugs that reduce, inhibit, block, interfere with or interfere with the binding of leptin to the leptin receptor or its extracellular domain; antagonists of signals that govern leptin production or synthesis. Drugs and drugs that inhibit leptin from reaching the leptin receptor (eg, block leptin from crossing the blood brain barrier). Such an agent may be any organic molecule that inhibits or prevents the interaction of leptin and the leptin receptor or leptin production. See U.S. Pat. No. 5,866,547.

As used herein, a leptin receptor agonist refers to a factor that activates, induces, or increases the action of a leptin receptor. Such agonists may include compounds that bind leptin or bind leptin receptors. The antagonist is an output of the leptin receptor, that is, an intracellular step of the leptin signal pathway following binding of leptin to the leptin receptor, that is, a downstream phenomenon that acts on the leptin / leptin receptor signal. Also included are compounds that activate, induce, or increase a phenomenon that does not occur at the level of ligand interaction. Leptin receptor agonists are proteins, antibodies, small organic molecules, or carbohydrates,
For example, but not limited to, leptin, leptin analogs, and antibodies that specifically bind and activate leptin. Leptin antagonists include, but are not limited to, anti-leptin antibodies, receptor molecules, derivatives that specifically bind to leptin and inhibit binding of leptin to its cognate receptor.

In addition, the Ob-binding protein is an inter-α-trypsin inhibitory heavy chain protein (I
HRP); α2-macroglobulin; and Ob-BP1 which can specifically bind to Ob and prevent Ob from binding to ObR, but are not limited thereto. Specific Ob binding proteins further allow for modulation of free Ob levels, immobilization, binding / free leptin assay. US Patent No. 6,919,902; Birkenmeire et al., 1998, Eu
r. J. Endocrin., 139: 224-230; Patel et al., 1999, J. Biol. Chem., 32: 22729-22.
See 738. Other Ob-binding proteins such as apolipoprotein are described in US Pat.
, 450.

An example of an ObR antagonist is acetylphenol, which is known to be useful as an antiobesity and antidiabetic compound. Since acetylphenols are antagonists of ObR, they prevent Ob from binding to ObR. Thus, in light of the teachings of the present invention, the compounds will effectively cause an increase in bone disease. See US Pat. No. 5,859,051 for specific structures of acetylphenols that can be used as ObR antagonists.

Further antagonists and agonists of ObR, as well as other compounds that modulate ObR gene expression or ObR activity, can be used for diagnosis, drug screening, clinical trial monitoring, and / or treatment of bone disorders. See US Pat. Nos. 5,972,621, 5,874,535, 5,912,123.

5.5 Methods for the Treatment or Prevention of Bone Diseases Bone diseases that can be treated and / or prevented according to the present invention include bone diseases characterized by reduced bone mass relative to bone mass of the corresponding healthy bone, osteoporosis, Not limited to osteopenia and Paget's disease. Bone disorders that can be treated and / or prevented according to the present invention also include bone disorders characterized by increased bone mass relative to the bone mass of the corresponding healthy bone, and are not limited to marble bone disease, osteosclerosis, osteochondrosis.

In one embodiment of the invention, a method of treating bone disorders comprises administering a therapeutically effective amount of a compound that reduces serum leptin levels to a mammal in need thereof. Is characterized by the reduced bone mass relative to the bone mass of the corresponding healthy bone. Some specific embodiments of those compounds and methods are not limited to those that inhibit or reduce leptin synthesis or increase leptin breakdown.
Among such compounds are the antisense, ribozyme or triple helix sequences of leptin-encoding polypeptides.

According to another aspect of the present invention, a bone disease treatment characterized in that a therapeutically effective amount of a compound that reduces leptin levels in cerebrospinal fluid is administered to a mammal in need thereof. There is a method and is characterized by a reduced bone mass relative to the bone mass of healthy bone to which the bone disease corresponds. Some specific embodiments of those compounds and methods are not limited to those that inhibit or reduce leptin synthesis or increase leptin breakdown, and compounds that bind leptin in blood.

Specific embodiments of the methods of the invention include, for example, a method of treating a bone disorder, which method comprises administering a compound in a therapeutically effective amount to a mammal in need thereof. Characteristically, characterized by a reduced bone mass relative to the bone mass of a healthy bone to which the bone disease corresponds,
The compound is selected from the group consisting of compounds that bind leptin in blood, but the compound is an antibody that specifically binds leptin, a soluble leptin receptor polypeptide, an inter-alpha-trypsin inhibitor heavy chain-related protein, alpha2 macroglobulin. It is not limited to compounds such as proteins.

According to another aspect of the present invention, a bone disorder characterized in that a compound that reduces the level of phosphorylated Stat3 polypeptide is administered in a therapeutically effective amount to a mammal in need thereof. There are treatment methods and is characterized by a reduced bone mass relative to the bone mass of healthy bone to which the bone disease corresponds. Some specific embodiments of those compounds and methods are specific to those that inhibit or reduce leptin synthesis or increase leptin breakdown, compounds that bind leptin in blood, and acetylphenol compounds, leptin. Binding to antibodies, antibodies that specifically bind to leptin receptors, leptin receptor antagonist compounds such as compounds such as soluble leptin receptor polypeptide sequences.

According to another aspect of the invention, a bone characterized in that a compound which reduces the level of leptin receptors in the hypothalamus is administered in a therapeutically effective amount to a mammal in need thereof. There are methods of treating the disease, which are characterized by a reduced bone mass relative to the bone mass of the non-diseased bone to which the bone disease corresponds. Some specific embodiments of those compounds and methods are not limited to those that inhibit or reduce leptin receptor synthesis or increase leptin receptor breakdown. Among such compounds are the antisense, ribozyme or triple helix sequences of leptin-encoding polypeptides.

Compounds that reduce leptin levels in serum or cerebrospinal fluid are those that reduce leptin levels in the following assays. Leptin-expressing cell lines, preferably
Compounds are contacted with cells from the NIH3T3L1 cell line and it is determined whether leptin expression and / or leptin synthesis was reduced relative to the levels exhibited by the cell line in the absence of compound. Standard assays such as Northern blots can be used to measure levels of leptin expression and Western blots can be used to measure levels of leptin synthesis. An alternative assay compares leptin levels in mammals undergoing treatment for bone disease before and after administration of the compound, such that if leptin levels are reduced, the compound reduces leptin levels It is a thing. Similarly, compounds that increase leptin levels in serum or cerebrospinal fluid are those that increase leptin levels via such an assay.

Compounds that reduce the level of phosphorylated stat3 polypeptide, its downstream effector of leptin signaling in its target cells (Tartaglia et al., 1995, Cell 83,
1263-1271; Baumann et al., 1996, Proc Natl Acad Sci USA 93,8374-8378; Gbila
rdi et al., 1996, Proc Nati Acad Sci USA 93,6231-6235; Vaisse et al., 1996,
Nat Genet 14,95-97) reduces the level of phosphorylated stat3 in the following assay. Leptin polypeptides and compounds are contacted with cells expressing a functional leptin receptor and the level of phosphorylated stat3 polypeptide in the cells is measured. To measure the level of phosphorylation of stat3 polypeptide, the cells can be, for example, lysed and an appropriate assay (eg, Western blot) performed. If the level of phosphorylated stat3 is reduced relative to the level exhibited by the cell line in the absence of the compound, the compound is one that reduces the level of phosphorylated stat3. Similarly, a compound that increases levels of phosphorylated stat3 polypeptide in serum or cerebrospinal fluid is one that increases leptin levels via such an assay.

A compound that is said to be administered in a "therapeutically effective amount" is said to be in response to, for example, a disease condition if the amount administered results in the desired change in physiological function of the recipient mammal. If the result is an increase or decrease in bone mass relative to the bone mass of the bone to be treated, i.e., in the treatment, i.e. disease-free condition, the corresponding non-diseased bone (i.e. long bones, vertebrae, etc. The result is that the bone mass is adjusted to be more similar to the bone mass of the corresponding bone. Regarding those methods,
The corresponding non-diseased bone is the same type of bone as the bone being treated (eg, the corresponding vertebra or long bone), and bone mass is measured using standard techniques described above that are well known to those of skill in the art. The techniques include, for example, x-ray of bone mass, DEXA and classical histological diagnosis and measurement.

Among the compounds that can be utilized as part of the methods set forth herein are also those set forth herein, eg, those described and taught in the sections. , Compounds identified through techniques such as those described and taught in the Sections.

Specific techniques and methods that can be used as part of the therapeutic and prophylactic methods of the invention are detailed below.

5.5.1. Inhibition of Ob or ObR expression, level or activity to treat bone disease by increasing bone mass Ob or ObR expression (either transcription or translation), neutralizing or delaying level or activity Alternatively, any method of inhibiting can be used to treat or prevent a bone disease characterized by a decrease in bone mass relative to the corresponding non-diseased bone by achieving an increase in bone mass. Such an approach can be used for the treatment or prevention of bone diseases such as osteoporosis, osteopenia, defective bone formation or resorption, osteoarthritis, bone metastases. Such methods can be used to treat conditions involving fractures and damaged bone.

For example, compounds such as soluble peptides, proteins, fusion proteins, or antibodies (including anti-idiotype antibodies) that bind circulating Ob, a natural ligand for ObR and “neutralize” the natural ligand for circulating Ob, ObR. Can be used to achieve increased bone mass. Similarly, soluble peptides, proteins, fusion proteins, or antibodies (
Compounds such as (including anti-idiotypic antibodies) can be used to achieve increased bone mass. For this purpose, either a peptide corresponding to the ECD of ObR, a soluble deletion mutant of ObR, or their ObR domain or mutant fused to another polypeptide (eg IgFc polypeptide) is available. Is. Alternatively, anti-idiotype antibodies or Fab fragments of anti-idiotype antibodies that mimic ObRRED and neutralize Ob can be used. Alternatively, compounds that inhibit ObR homodimerization such that the affinity of leptin for the leptin receptor is reduced can be used. Devos et al., 1997, JBC, 272: 18304-18310. For treatment, such ObR peptides, proteins, fusion proteins, anti-idiotypic antibodies or Fabs are administered to a subject in need of treatment at therapeutically effective levels. For prophylaxis, such ObR peptides, proteins, fusion proteins, anti-idiotypic antibodies or Fabs are administered to subjects at risk of bone disease for a time and at a concentration sufficient to prevent bone disease.

In an alternative embodiment for neutralizing circulating Ob, cells genetically engineered to express such soluble or secreted forms of ObR can be administered to a patient. As a result, they act as a "bioreactor" in vivo, providing a continuous supply of Ob neutralizing proteins. Such cells can be obtained from patients or MHC matched donors, but are not limited to fibroblasts, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelial cells and the like. The cells are genetically engineered in vitro to use recombinant DNA technology, for example (using viral vectors, and preferably vectors which integrate the transgene into the cell's genome)
For ObRECD, or ObR-Ig, depending on transfer or transfection procedure
The coding sequence for the fusion protein is introduced into the cells, but the plasmid, cosmid, YA
It is not limited to the use of C electroporation and liposomes. The ObR coding sequence can be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer, which effects expression and secretion of the ObR peptide or fusion protein. Engineered cells expressing and secreting the desired ObR product can be introduced systemically into the patient, for example, in the blood circulation, intraperitoneally, choroid plexus or hypothalamus. Alternatively, the cells can be incorporated into a matrix and transplanted into the body, for example, genetically engineered fibroblasts can be transplanted as part of the skin graft,
Genetically engineered endothelial cells can be transplanted as part of a blood vessel substitute. (
See, for example, Anderson et al., US Pat. No. 5,399,349 and Mulligan & Wilson, US Pat. No. 5,460,959. Each is incorporated herein by reference in its entirety. 3.) When the cells to be administered are non-autologous, they can be administered using known techniques to prevent the development of a host immune response against the introduced cells.
For example, cells can be introduced in an encapsulated form, which allows exchange of components in the immediate extracellular environment, allowing the introduced cells to be recognized by the host immune system. I don't.

In an alternative embodiment, bone disease treatments can be designed to reduce the level of endogenous Ob or ObR gene expression, eg, translation of Ob or ObR mRNA transcripts using an antisense or ribozyme approach. To prevent or prevent
Inhibit or "knock out" the Ob or ObR gene, or its endogenous promoter, using the helix approach to inhibit transcription of the Ob or ObR gene, or use targeted homologous recombination . The ObR gene is expressed in the brain, including the choroid plexus and hypothalamus, and the delivery technology is preferably one designed to cross the blood-brain barrier (see WO89 / 10134, which is hereby incorporated by reference in its entirety). Incorporated into the book). Alternatively, the antisense, ribozyme or DNA constructs described herein can be administered directly to the site containing the target cells, such as the choroid plexus, hypothalamus, adipose tissue and the like.

The antisense approach involves the design of oligonucleotides (either DNA or RNA) that are complementary to Ob or ObR mRNA. Antisense oligonucleotides will bind to complementary Ob or ObR mRNA transcripts and prevent translation.
Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of RNA, as referred to herein, means a sequence that has sufficient complementarity to be capable of hybridizing with RNA and forms a stable duplex. . In the case of double-stranded antisense nucleic acids, single strands of duplex DNA can thus be tested or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the hybridized nucleic acids, the more bases will mismatch with the RNA, including the stable duplex (or triplex in some cases) and still form. Those skilled in the art
Standard procedures can be used to determine the acceptable degree of mismatch by measuring the melting point of the hybrid complex.

Those skilled in the art will appreciate that the regulation of gene expression will design antisense molecules in the control regions of the leptin or leptin receptor genes, ie promoters, enhancers, and introns, as well as the coding regions of those genes. Recognize that can be earned by. Such sequences are referred to herein as leptin-encoding polynucleotides or leptin receptor-encoding polynucleotides, respectively.

Oligonucleotides derived from the transcription start site, eg between the -10 and +10 regions of the leader sequence, are preferred. Oligonucleotides that are complementary to the 5'end of the message, eg, up to and including the AUG start codon, and 5'untranslated sequences, generally work most effectively at inhibiting translation. However, sequences complementary to the 3'untranslated sequence of mRNA have recently been shown to be effective in inhibiting translation of mRNA. See generally Wagner, R., 1994, Nature 372: 333-335. Oligonucleotides complementary to the 5'untranslated region of the mRNA include the complement of the AUG start codon. Antisense oligonucleotides complementary to the MRNA coding region are less effective inhibitors of translation but can be used according to the invention. Antisense nucleic acids should be at least 6 nucleotides in length, even if designed to hybridize to the 5 ', 3'or coding regions of the Ob or ObR mRNA.
Preferred are oligonucleotides ranging in length from 6 to about 50 nucleotides.
In a particular embodiment the oligonucleotide is at least two or more nucleotides,
At least 17 nucleotides, at least 25 nucleotides or at least 5
It is 0 nucleotides.

Regardless of the choice of target sequence, in vitro studies are preferably performed first to quantify the ability of antisense oligonucleotides to inhibit gene expression.
It is preferred that those studies use controls that discriminate between antisense gene inhibition and non-specific biological effects of oligonucleotides. It is also desirable that those studies compare the levels of target RNA or protein to the levels of internal control RNA or protein. In addition, it is envisioned that the results obtained using the antisense oligonucleotide are compared to the results obtained using the control oligonucleotide. It is preferred that the control oligonucleotide is about the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence only as necessary to prevent specific hybridization to the target sequence.

Oligonucleotides can be DNA or RNA or chimeric mixtures, or derivatives, or modified versions thereof, single-stranded or double-stranded. Oligonucleotides can be modified with base moieties, carbohydrate moieties, or phosphate backbones, for example to improve stability of the molecule, hybridization, etc. Oligonucleotides can be peptides (eg, for targeting host cell receptors in vivo), or agents that facilitate transport across cell membranes (eg, Letsinger et al., 1989, Proc. Na.
tl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc. Nathl. A
cad. Sci. 84: 648-652; PCT application No. WO88 / 09810, Dec.15, 1988), or blood-brain barrier (see PCT application No. WO89 / 10134, Apr.25, 1988), Resolving agents triggered by hybrid formation (eg Krol et al., 1988, Bio Techniques 6: 958-9
76)), or other accessory groups such as intercalating agents (see, eg, Zin, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide can be conjugated to another molecule such as, for example, a peptide, a hybridisation-triggered cross-linking agent, a transport agent, a hybridisation-triggered resolving agent.

Antisense oligonucleotides can include at least one modified base moiety, which base moiety is 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine. , 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosequeosin, inosine, N6-iso Pentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 -Methylaminomethyluracil, 5-methoxyaminomethyl
-2-thiouracil, β-D-mannosyleosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil , Queocin,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxoacetic acid (v), 5-methyl-2- Thiouracil, 3- (3-amino-3-N-2-carboxypropyl
) Uracil, (acp3) w, selected from the group not limited to 2,6-diaminopurine.

Antisense oligonucleotides can also include at least one modified carbohydrate moiety selected from the group not limited to arabinose, 2-fluoroarabinose, xylulose, hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone, wherein the phosphate backbone is phosphorothioate, phosphorodithioate, phosphoramidioate, phospho. It is selected from the group consisting of luamidate, phosphoramidiate, methylphosphonate, alkylphosphoester, formacetal and the like.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. α-anomeric oligonucleotide is a complementary RNA
, The strands run parallel to each other as opposed to the normal β-unit (Grautier et
al., 1987, Nucl. Acides Res. 15: 6625-6641), forming specific double-stranded hybrids. Oligonucleotides are 2'-0-methyl ribonucleotides (Inoue et al., 19
87, Nucl. Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analog (Inoue e
t al., 1987, FEBS Lett. 215: 327-330).

The oligonucleotides of the invention can be synthesized by well-known standard methods, eg, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). As an example, phosphorothionate oligonucleotides are described by Stein et al. (1988, Nucl. Acids Res. 16: 3209).
The methylphosphonate oligonucleotides can be synthesized by a porous glass polymer support (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85
: 7448-7451) and so on.

While antisense nucleotides complementary to the Ob or Ob coding region sequences can be used, those complementary to the transcribed untranslated region are most preferred. For example, an antisense oligonucleotide for the ObR coding region having the following sequence,
It can be used according to the invention.

A) 5'-CATCTTACTTCAGAGAA-3 'b) 5'-CATCTTACTTCAGAGAAGTACAC-3' c) 5'-CATCTTACTTCAGAGAAGTACACCCATAA-3 'd) 5'-CATCTTACTTCAGAGAAGTACACCCATAATCCTCT-3' e) 5'-AATCATCTTACTTCAGAGAAGTACACCCATAAT) 5'-CTTACTTCAGAGAAGTACACCCATAATCC-3 'g) 5'-TCAGAGAAGTACACCCATAATCC-3' h) 5'-AAGTACACCCATAATCC-3 'Antisense molecules can deliver Ob or ObR to cells expressing in vivo. Numerous methods have been developed to deliver antisense DNA or RNA to cells, for example, the antisense molecule can be directly injected into a tissue site, or a modified antisense molecule, to target the desired cell (eg, target). Antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the cell surface)
Can be designed to be targeted and can be administered systemically.

A preferred approach to achieve intracellular concentrations of antisense sufficient to prevent translation of endogenous mRNA is that the antisense oligonucleotide is a strong pol
Utilize a recombinant DNA construct placed under the control of the III or Pol II promoter. The use of such constructs to transfect target cells in a patient is
Single-stranded RNA that will form complementary base pairs using an endogenous Ob or ObR transcript
Results in a sufficient amount of transcription and thus prevents translation of the Ob or ObR mRNA, respectively. For example, the vector can be introduced in vivo so that it is taken up by the cell and directs transcription of the antisense RNA. Such vectors can remain episomal or become chromosomally integrated, as long as they can be transcribed to produce the desired antisense RNA. Such vectors can be made by recombinant DNA technology methods standard to those of skill in the art.
Vectors can be plasmids, viruses, or others known in the art and can be used for replication and expression in mammalian cells. Expression of the antisense RNA-encoding sequence is expressed in order to operate in mammals, preferably human cells,
It can be by any known promoter. Such promoters can be inducible or constitutive. One such promoter is SV40
Early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310)
, A promoter contained within the 3'long terminal repeat of Rous sarcoma virus (Yamamoto
et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Nathl. Acad. Sci. USA 78: 1441-1445),
Regulatory sequence of metallothionein gene (Brinster et al., 1982, Nature 296: 39-
42), and so on. Any type of plasmid, cosmid, YAC or viral vector can be used for the preparation of recombinant DNA constructs, which should be introduced directly into the tissue site, eg choroid plexus, hypothalamus. You can Alternatively, a viral vector can be used, which selectively infects the desired tissue (eg, for the brain, a herpesvirus vector can be used), in which case administration is by another route (eg, systemic). Can be accomplished in.

Ribozyme molecules designed to catalytically cleave Ob or ObR mRNA transcripts can also be used to prevent transcription of Ob or ObR mRNA and expression of Ob or ObR. (For example, PCT international application WO90 / 11364, Oct. 4, 1990; Sarver
et al., 1990, Science 247: 1222-1225). Ribozymes that cleave mRNAs at site-specific recognition sequences can be used to disrupt Ob or ObR mRNAs, although hammerhead ribozymes are preferred. Hammerhead ribozyme
At a position dependent on flanking regions that form complementary base pairs with the target mRNA
Split the mRNA. The only requirement is that the target mRNA has the following sequence of two bases 5'-UG-3 '. The construction and production of hammerhead ribozymes are well known and are more fully described in Haseloff and Gerlach, 1988, Nature, 334: 585-591. There are hundreds of potential hammerhead ribozyme cleavage sites within the nucleotide sequence of human Ob and ObR cDNAs. See, for example, US Pat. No. 5,972,621. Preferably, the ribozyme is designed such that the split recognition site is located near the 5'end of the Ob or ObR mRNA, ie, it increases efficiency and is non-functional.
Minimize intracellular accumulation of mRNA transcripts.

For example, a hammerhead ribozyme directed against ObR mRNA having the following sequence can be utilized in accordance with the invention.

[0217] a) 5'ACAGAAUUUUUGACAAAUCAAAGCAGANNNNUCUGAGNAGUCCUUACUUCAGAGAA-3 ', b) 5'-GGCCCGGGCAGCCUGCCCAAAGCCGGNNNNCCGGAGNAGUCGCCAGACCGGCUCGUG-3', c) 5'-UGGCAUGCAAGACAAAGCAGGNNNNCCUGAGNAGUCCUUAAAUCUCCAAGGAGUAA-3 ', d) 5'-UAUAUGACAAAGCUGUNNNNACAGAGNAGUCCUUGUGUGGUAAAGACACG-3', e) 5'-AGCACCAAUUGAAUUGAUGGCCAAAGCGGGNNNNCCCGAGNAGUCAACCGUAACAGUAUGU-3 ', f) 5'-UGAAAUUGUUUCAGGCUCCAAAGCCGGNNNNCCGGAGNAGUCAAGAAGAGGACCACAUGUCACUGAU
GC-3 ', g) 5'-GGUUUCUUCAGUGAAAUUACACAAAGCAGCNNNNGCUGAGNAGUCAGUUAGGUCACACAUC-3', h) 5'-ACCCAUUAUAACACAAAGCUGANNNNUCAGAGNAGUCAUCUGAAGGUUUCUUC-3 '. The ribozyme of the present invention is known as (IVS) or LIV RNA, or IVS or L-RNA in the IVS or L-RNA. And has been extensively described by Thomas Check and coworkers (Zaug et al., 1984, Science, 224: 574-578;
Zaug and Cech, 1986, Science, 231: 470-475; Zaug et al., 1986, Nature, 32.
4: 429-433; published international patent application WO88 / 04300 University Patents Inc .; Been and Cech, 1986, Cell, 47: 207-216)), and RNA endoribonuclease (hereinafter "check ribozyme"). The check ribozyme has an eight base pair active site, which site is congested with the target RNA sequence, followed by division of the target RNA. The invention includes those checked ribozymes that target the eight base pair active site sequences shown in Ob and ObR.

As in the antisense approach, ribozymes can be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) and can be expressed in cells expressing Ob and ObR in vivo. Will be delivered. A preferred method of delivery comprises using a DNA construct encoding a ribozyme under the control of a strong constitutive Pol III or Pol II promoter,
Thus, the transfected cells will produce sufficient quantities of ribozymes to disrupt the endogenous Ob or ObR message and suppress translation. Unlike antisense molecules, ribozymes are catalytic, so low intracellular concentrations are required for efficacy.

Similarly, leptin or leptin receptor inhibition can be accomplished using a "triple helix" base pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
Techniques for utilizing the triple helix technique are well known to those of skill in the art. Genera
lly Helene (1991) Anticancer Drug Des. 6 (6): 569-84; Helene (1992) Ann.
See NY Acad. Sci. 660: 27-36; and Maher (1992) Bioassays 14 (12): 807-15.

Endogenous Ob or ObR gene expression was observed using targeted homologous recombination in Ob.
Alternatively, it can be reduced by inactivating or "knocking out" the ObR gene or its promoter. (For example, Smithies et al., 1985,
Nature 317: 230-234; Thomas & Capecchi, 1987, Cell 51: 503-512; Thompso
n et al., 1989 Cell 5: 313-321; they are all included by reference). For example, a mutant, non-functional Ob flanked by endogenous DNA or homologous ObR genes (either coding or regulatory regions) by DNA homology.
Or ObR (or a completely unrelated DNA sequence) is used to transfect cells expressing Ob or ObR in vivo with or without a selectable marker and / or a negative selectable marker. be able to. Insertion of the DNA construct results in inactivation of the Ob or ObR gene via targeted homologous recombination. Such an approach is particularly suitable for the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal progeny with inactive ObR (eg Thomas & Capecchi 1987).
and Thompson 1989, supra). However, this approach uses a suitable viral vector, such as a herpesvirus vector for delivery to brain tissue, such as the hypothalamus, choroid plexus, or adipose tissue, at the site where the recombinant DNA construct is required. If administered or targeted directly in vivo, it can be adapted for human use.

Alternatively, the endogenous Ob or ObR gene expression is altered by the Ob or ObR gene (
That is, a triple helix structure that can be reduced by targeting a deoxyribonucleotide sequence complementary to the control region of the promoter and / or enhancer) and prevents transcription of the Ob or ObR gene in the target cells of the body. Form. (Generally, Helene, C. 1991, Anticancer Drug Des., 6 (6): 569-84; He
lene, C., et al., 1992, Ann, NY Accad. Sci., 660: 27-36; and Maher, L.
J., 1992, Bioassays 14 (12): 807-15).

In yet another embodiment of the invention, the activity of Ob or ObR can be reduced using a “domain negative” approach to achieve increased bone mass.
Because of this, constructs encoding defective Ob or ObR can be used in gene therapy approaches, reducing the activity of Ob or ObR in appropriate target cells.

For example, a nucleotide sequence directing host cell expression of ObR in which CD or a portion of CD has been deleted or mutated may be used in the choroid plexus or thalamus (by either in vivo or ex vivo gene therapy methods described above). It can be introduced into the cells below. Alternatively, targeted homologous recombination can be used to introduce such deletions or mutations into the endogenous ObR gene of the hypothalamus or choroid plexus of the subject. The engineered cells will express a non-functional receptor (ie, a fixed receptor that is able to bind its natural ligand, but not signal transduce). Such engineered cells shown in the choroid plexus or in the hypothalamus demonstrate a diminished response to endogenous Ob ligand, resulting in increased bone mass.

An additional embodiment of the invention is a method of reducing leptin levels by increasing the disruption of the leptin protein, ie by binding the antibody in such a way as to target the leptin protein for elimination. Another embodiment of the invention is a method of reducing the leptin receptor by increasing the disruption of the leptin receptor protein, ie by binding the antibody in such a way as to target the leptin receptor protein for elimination. . Another embodiment consists in reducing leptin levels by increasing the synthesis of the soluble form of the leptin receptor which binds free leptin.

Another embodiment of the invention is a method of administering a compound that affects the structure, function or homodimerization properties of the leptin receptor. Such compounds include proteins, nucleic acids, carbohydrates or other molecules that modify the structure, function or homodimerization properties of the leptin receptor at the time of binding, thus rendering the receptor ineffective in its activity, I'm not limited to them.

5.5.2. Restoring or Increasing Ob or ObR Expression or Activity to Reduce Bone Mass For the treatment of bone disorders with respect to increased levels of normal Ob or ObR gene expression and / or gene product activity. Ob or ObR nucleic acid sequences can be utilized. If the cause of the disorder is a defect in Ob or ObR, then treatment can be given, for example in the form of gene replacement therapy. Specifically, one or more copies of the normal Ob or ObR gene or a portion of the Ob or ObR gene that induces the production of a normal functioning Ob or ObR gene product is introduced into intracellular DNA, such as liposomes.
In addition to other particles to be introduced, vectors including, but not limited to, adenovirus, adeno-associated virus, retrovirus and herpesvirus vectors can be used to It can be inserted into cells.

Since the ObR gene is expressed in the brain, including the choroid plexus and hypothalamus, such replacement therapy techniques involving ObR have led to these cell types in the patient being obR
It should be possible to deliver the gene sequence. Thus, techniques for delivery of the ObR gene sequence should be designed to readily cross the blood-brain barrier, as is well known to those of skill in the art (eg, incorporated herein by reference in its entirety). PCT Application No. WO 89/10134), or alternatively such Ob to a site in the cell where the ObR gene sequence is to be expressed.
Direct administration of the R gene sequence should be involved. Alternatively, targeted homologous recombination can be utilized to correct a defect in the endogenous Ob or ObR gene in the tissue of interest. In animals, targeted homologous recombination can be used to correct defects in ES cells to produce progeny with the corrected trait.

Additional methods available for increasing the overall level of expression and / or activity of the Ob or ObR gene include sufficient numbers to ameliorate the symptoms of bone disorders associated with increased bone mass. And introducing the appropriate Ob or ObR-expressing cells into the patient at the location. Such cells may be recombinant or non-recombinant. Cells that can be administered to increase the overall level of Ob or ObR gene expression in a patient include normal cells, preferably choroid reticulum cells or hypothalamic cells expressing the ObR gene or adipocytes expressing the Ob gene. There is. The cells may be administered at an anatomical site within the brain or adipose tissue, or alternatively as part of a tissue graft located at a different site within the body. Such cell-based gene therapy techniques are well known to those of ordinary skill in the art. For example, Ande
See rson, et al., US Pat. No. 5,399,349; Mulligan & Wilson, US Pat. No. 5,460,959.

Finally, in order to achieve a reduction in bone mass, activation of an ObR by, for example, activating a downstream signaling protein in the ObR cascade and thus bypassing the ObR defect is performed. The compounds identified in the above assay which stimulate or enhance the introduced signal can be used.
The formulation and mode of administration will depend on the physicochemical properties of the compound. Administration should include known techniques that allow for crossing the blood-brain barrier.

5.5.3. Gene Therapy Approach to Regulation of Ob and ObR Activities and Treatment or Prevention of Bone Disorders Expression of Ob and ObR uses gene therapy approaches to regulate Ob and ObR activity and treat bone disorders. (Eg, at the transcriptional or translational level) can be regulated in vivo. Several approaches are described below.

Regarding increased levels of normal Ob and ObR gene expression and / or Ob and ObR gene product activity, Ob and ObR nucleic acid sequences can be utilized for the treatment of bone disorders. If the cause of the bone disease is a defect in Ob or ObR, the treatment can be carried out, for example, in the form of gene replacement therapy. Specifically, normal Ob or ObR
Adenovirus, adeno-associated virus, in addition to other particles that introduce DNA into cells, such as liposomes, one or more copies of the gene or a portion of the gene that induces the production of a gene product that exhibits normal function, Vectors, including (but not limited to) retroviruses and herpesviruses, can be used to insert into appropriate cells within the body of patients and test animals.

Since the ObR gene is expressed in the brain, including the cortex, thalamus, brainstem and spinal cord, and the hypothalamus, such replacement therapy techniques have shown that these cell types in the patient's body have ObR
It should be possible to deliver the gene sequence. Thus, techniques for delivery of ObR gene sequences should be designed to readily cross the blood-brain barrier, as is well known to those of skill in the art (eg, incorporated herein by reference in its entirety). See PCT Application No. WO 89/10134), which is otherwise alternatively such O to the site of the cell where the ObR gene sequence is to be expressed.
Direct administration of the bR gene sequence should be involved.

Alternatively, targeted homologous recombination can be utilized to correct defects in the endogenous Ob or ObR genes in relevant tissues, such as adipose and brain tissues, respectively. In animals, targeted homologous recombination can be used to correct defects in ES cells to produce progeny with the corrected trait.

Additional methods available to increase the overall level of Ob and ObR gene expression and / or activity include osteopetrosis, osteosclerosis and osteomalacia (
(But not limited to) including the introduction of appropriate Ob or ObR expressing cells into the body of the patient in sufficient number and location to ameliorate the symptoms of the bone disorder. Such cells may be recombinant or non-recombinant. Cells that can be administered to increase the overall level of Ob or ObR gene expression in a patient's body include normal cells or adipose or hypothalamic cells expressing the Ob or ObR gene, respectively. The cells may be administered at an anatomical site in adipose tissue or in the brain, or alternatively as part of a tissue graft located at a different site in the body. Such cell-based gene therapy techniques are well known to those of ordinary skill in the art. For example, Anderson, et al., US Pat. No. 5,399,349; M
See ulligan & Wilson, US Pat. No. 5,460,959.

5.6 Pharmaceutical Formulations and Methods for the Treatment of Bone Disorders The compounds of the present invention inhibit various bone disease states by any means that result in the contact of the active ingredient with the site of action of its agent in the body of the mammal. Can be administered and administered. These compounds can be administered either as individual therapeutically active ingredients or in the form of combinations of therapeutically active ingredients by any conventional means available for combined use with pharmaceuticals. They may be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dose administered will be a therapeutically effective amount of the compound sufficient to result in amelioration of the symptoms of bone disease, and, of course, the pharmacodynamic properties of the particular active ingredient and its mode and route of administration; It will vary depending on known factors such as the age, sex, health and volume of the recipient; the nature and extent of the symptoms; type of co-treatment, frequency of treatment and desired effect.

5.6.1. Dose Determination The toxicity and therapeutic efficacy of such compounds can be determined, for example, by the LD ₅₀ (lethal dose for 50% of the population) and ED ₅₀ (therapeutically effective dose in 50% of the population). To determine, it is determined by standard pharmaceutical procedures in cell cultures or laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED _50. Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects can also be used, but to design a delivery system that targets such compounds to the site of diseased tissue in order to minimize the potential for damage to uninfected cells and reduce side effects. You should pay attention to.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dose of such compound is preferably
It is within the range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose is
It can vary within this range depending on the dosage form utilized 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. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ as determined in cell culture (ie, the concentration of test compound that can inhibit half the maximal level of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Specific doses may be utilized for the antibody. Typically, the preferred dose is 0.1 mg to 100 mg / kg body weight (generally 10 mg / kg to 20 mg).
/ Kg), usually 50 mg / kg to 100 if the antibody is to act in the brain
A dose of mg / kg is appropriate. If the antibody is a partially human antibody or a fully human antibody, it will have a longer half-life in the human body than other antibodies.
Thus, lower doses are often possible with partially human and fully human antibodies. Additional modifications can be used to further stabilize the antibody. Lipidation can be used, for example, to stabilize antibodies and enhance uptake and tissue penetration (eg, into the brain). Cruikshank e is a method for the fatification of antibodies.
t al. ((1997) J. Acquired immunodeficiency syndrome and human retrovirology 14; 1
93).

A therapeutically effective amount of protein and polypeptide (ie, an effective dose) is about 0.001-30 mg / kg body weight, preferably about 0.01-25 mg, more preferably about 0.1-20 mg. And more preferably about 1 to 10 mg, 2 to 9 mg, 3 to
It ranges from 8 mg, 4-7 mg or 5-6 mg.

Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody may include a single treatment or, preferably, includes a series of treatments. It may be. In a preferred example, the subject is about 0.1 per kg body weight.
In the range of about 20 mg, about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks and even more preferably about 4,5 or 6 weeks, once a week, Receive antibody, protein or polypeptide therapy.

The present invention further includes agents that modulate expression or activity. The agent may be a small molecule. For example, such small molecules include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic with a molecular weight of less than about 10,000 grams per mole. Compounds (ie including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, 1 It includes, but is not limited to, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole and salts, esters and other pharmaceutically acceptable forms of such compounds.

It will be appreciated that the appropriate dosage of a small molecule agent will depend on a number of factors known to those of ordinary skill in the art, such as a physician. The small molecule dose will depend, for example, on the identity, size and conditions of the subject or specimen to be treated, and, where applicable, the route by which the composition is to be administered and the small molecule is a nucleic acid or polypeptide of the invention. It will vary depending on the effect the doctor wants to have. Examples of dosages include milligram or microgram amounts of small molecules per kg body weight or specimen weight, eg, about 1 microgram to about 500 milligrams per kilogram, about 100 micrograms to about 5 milligrams or about 1 microgram to about 1 microgram. Approximately 50 micrograms are included.

5.6.2 Formulation Forms and Uses Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner with one or more physiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts and solvates are formulated for administration by inhalation or insufflation (either through the mouth or nose) or orally, sublingually, parenterally or rectally. be able to.

For oral administration, the pharmaceutical composition may be eg a binder (eg pre-gelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg lactose, microcrystalline cellulose or hydrogen phosphate). Calcium); lubricants (eg magnesium stearate, talc or silica) disintegrating agents (eg potato starch or sodium starch glycolate); or pharmaceutically acceptable excipients such as wetting agents (eg sodium lauryl sulphate). Can be used in the form of tablets or capsules prepared by conventional methods. The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form, for example, of solutions, syrups or suspensions, or dry product presentations adapted for constitution with water or other suitable vehicle before use. You can take it. Such a liquid preparation is
Suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible oils)
Pharmaceutical agents such as: emulsifiers (eg lecithin or gum arabic); non-aqueous vehicles (eg tonsil oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (eg methyl or propyl-p-hydroxybenzoate or sorbic acid) It can be prepared by conventional means with acceptable additives. The preparation may optionally contain buffer salts, flavoring agents, colorants and sweeteners.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For sublingual administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the invention may be provided with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is suitably used to be delivered in the form of an aerosol spray from a pressure pack or nebulizer. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of eg gelatin for use in an inhaler or insufflator may be formulated containing a suitable powder and compound powder mixture such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, eg by bolus injection or continuous infusion. Formulations for injection may be presented in unit dose form, such as ampoules or multi-dose containers, optionally with preservatives. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use. In general, water, suitable oils, aqueous dextrose (glucose) and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain an aqueous solution of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Also used are citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are Remington's Pharmaceut, a standard reference text in this field.
It is described in ical Science.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds can also be formulated as a depot preparation.
Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymers or hydrophobic materials (eg, as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, eg, low soluble salts. You can

In addition, standard pharmaceutical methods are available for controlling the duration of action. These are well known in the art and include release control preparations and are suitable such as, for example, polymers, polyesters, polyamino acids, polyvinyls, pyrrolidones, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. Macromolecules can be included. The concentration of macromolecules as well as the method of uptake can be adjusted to control release. In addition, the active substance
It can also be incorporated into particles of polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymers. These agents can also be used to entrap the compound within the microcapsules at the same time it is entrapped.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack can include plastic or metal foil, for example, a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

Useful pharmaceutical dosage forms for administration of the compounds of the invention can be illustrated as follows.

Capsules: Capsules are filled with standard two-piece hard gelatin capsules, each with the desired amount of powdered active ingredient, 175 mg lactose, 24 mg talc and 6 mg magnesium stearate. It is prepared by

Soft Gelatin Capsules: A mixture of active ingredients in soybean oil is prepared and injected into gelatin using a positive displacement pump to form soft gelatin capsules with the required amount of active ingredient. The capsules are then washed and dried.

Tablets: Tablets were prepared in dosage units in the desired amount of active ingredient, 0.2 mg colloidal silicon dioxide, 5 mg magnesium stearate, 275 mg microcrystalline cellulose, 11 mg corn starch and 98. Prepared by conventional procedures in the form of 8 milligrams of lactose. Appropriate coatings can be applied to increase palatability or delay absorption.

Injectables: A parenteral composition suitable for administration by infusion is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

Suspension; 100 milligrams of subdivided active ingredient every 5 millimeters, 2
Aqueous suspension for oral administration in such a form that it contains 00 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution U.S.P. and 0.025 mm of vanillin. Is prepared.

Gene Therapy Administration: If applicable, the gene therapy vector may be tablets, capsules,
Formulations in solid, semi-solid, liquid or gaseous form, such as powders, granules, ointments, solutions, suppositories, injectables, inhalants and aerosols, are formulated in the usual manner for their respective routes of administration. . Using means known in the art,
The release and absorption of the composition can be prevented or a timed release of the composition can be ensured until the target organ is reached. A pharmaceutically acceptable form that does not abrogate the compositions of the present invention should be utilized. In the pharmaceutical dosage form, the compositions can be used alone or in combination with other pharmaceutically active compounds in suitable combination as well as in combination.

Accordingly, the pharmaceutical compositions of the present invention can be delivered to various sites within the animal body via various routes to achieve particular effects. (Eg Rosenfeld
et al. (1991). Previous Rosenfeld et al., Clin. Res. 39 (2), 311
A (1991a); Jaffe et al., Supra, Berkner, supra). One of skill in the art will appreciate that although multiple routes of administration may be used, the specific route will be
It will be recognized that it can provide a more direct and effective response than the one pathway. Topical or systemic delivery is by application of the formulation into body cavities or by administration including instillation, inhalation or insufflation of aerosols or by parenteral introduction including intramuscular, intravenous, intraperitoneal, subcutaneous, cynic and topical application. Can be achieved.

The compositions of the present invention may be formulated in a predetermined amount of each dosage unit, eg, a teaspoonful, a tablet, a solution or a suppository, alone or in combination with other active agents as appropriate. It may be provided in a unit dose form such as containing the product. As used herein, "unit dosage form" means a predetermined amount, calculated in an amount sufficient to produce the desired effect, in association with the pharmaceutically acceptable diluent, carrier, or vehicle, where applicable. A physical discrete amount suitable as a unitary dose for human and animal subjects, each containing a quantity of the composition of the invention either alone or in combination with other active agents. It is a unit. The specifications for the unit dosage forms of the present invention will depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition within a particular host.

Accordingly, the present invention also preferably employs any of the routes of administration described above or alternative routes known to those of ordinary skill in the art and appropriate for the particular application, preferably in compositions. The present invention also provides a method of transferring a therapeutic gene to a host, which comprises administering the vector of the present invention as part of the above. An "effective amount" of a composition is that amount which produces a desired effect in the host, which can be monitored using multiple endpoints known to those of skill in the art. Effective gene transfer of a vector into a host cell according to the invention (to a host cell) may be in the form of a therapeutic effect (eg alleviation of symptoms associated with a particular disease to be treated) or additionally (in the host cell). Polymerase chain reaction in combination with sequencing, Northern or Southern hybridizations or transcription assays to detect the nucleic acid of, or affect levels or functions encoded by or transferred by the transferred nucleic acid. By expression of genes in the host or evidence of transferred genes (using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies or specialized assays to detect the received protein or polypeptide), Can be monitored.

The methods described herein are not all-inclusive and will be apparent to those of ordinary skill in the art additional methods to suit a particular application. Moreover, the effective dose of the composition can be further approximated through analogy to compounds known to exert the desired effect.

In addition, current dosages and schedules may vary depending on whether the composition is administered in combination with other pharmaceutical compositions or on individual differences in pharmacokinetics, drug transference and metabolism. . Similarly, the amount will depend on the particular cell line utilized (eg, the number of adenovirus receptors present on the cell surface or the particular vector utilized for gene transfer within that cell line). Can vary (based on ability to replicate). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted into the vector and the nature of the sequence, which is particularly empirically determined. Is a required parameter,
It may be modified due to factors that are not unique to the method of the invention (such as the costs associated with the synthesis). One of ordinary skill in the art can readily make any necessary adjustments according to the exigencies of the particular situation.

The following examples are provided by way of example and are not intended to limit the scope of the invention in any way.

6. Example 6.1. Animal Production, Characterization and Treatment Animal breeding and mutant mice (C57BL / 6J Lep ^ob , C from the Jackson Laboratory)
57BL / 6JLepr ^db , C57BL / 6JA ^Y / a) was purchased. A-ZI
The generation of P / FI transgenic mice has been previously reported (Moitra et.
al., 1998, Genes Dev 12, 3168-3181). Genotyping was performed according to established protocols. (Chua et al., 1997, Genomics 45, 2
64-270; Moitra et al., 1998, Genes Dev 12, 3168-3181.
Namac et al., 1998, Lab Animal Sci 48.103-104), animals had a standard diet (Purina # 5001) and, where indicated, a high fat / high carbohydrate diet (Bio-Serv #). F3282) was given. Bone specimens were processed as described (Ducy et al., 1999, Genes Dev 13.1025-1036).

6.2. Demonstration of Bone Mass Phenotype in ob / ob and db / db Mice Hypoactive dysfunction induces an increase in osteoclast number and bone resorption activity, which leads to a low bone mass phenotype (Riggs and Melton, 1986, N Engl J Med 314,167
6-1678). Thus, ob / ob mice with hypothalamic-derived hypogonadism should have lower bone mass than wild-type littermates (Ahima et al., 199).
6, Nature 382, 250-252; Chehab et al., 1996. Nat Genet 12
, 318-320; Ahima et al., 1997, J Clin Invest 99,391-3.
95). To determine whether obesity in ob / ob mice may affect its expected low bone mass phenotype, we used Faxitron (Phillips) to determine 6 months old wild type and X-ray analysis of the spine and long bones of ob / ob mice was performed. Surprisingly, the bones of ob / ob mice appeared to be much denser than that of their wild-type littermates (FIG. 1A), which indicated that a greater amount of mineralized bone matrix was present. I was proving. Increased bone density represented a significant change in bone architecture (ie, affecting more than 30% of the bone matrix) due to insensitivity to X-rays to quantify abnormal bone mass. .

Histological analysis was performed on undecalcified sections stained with von Kossa reagent and counterstained with Kernechtrot (Amling et al., 1999, Endocrin. 140: 4.
982-4987). In the 3 and 6-month-old mice, it was observed that the bones of the ob / ob mice had many more trabecular bones thicker than those of the wild-type mice (FIG. 1B). Cortical bone was unaffected. Osteomeasure Analy
Histomorphometric quantification according to standard techniques was performed using the sis System (Osteometrics, Atlanta) (Parfitt et al., 1987, J Bone Min Res 2, 59).
5-610). Statistical differences between groups (n = 4-6) were assessed by Student's test. Experiments showed that trabecular bone volume was increased approximately 2-fold in the long bones and spine of ob / ob mice compared to wild type littermates (FIG. 1c).
. This phenotype was observed in both genders. The functional consequences of this increase in bone mass are that wild-type mice (oV) were ovariectomized for 4 months to mimic the hypogonadal state of 6-month old ob / ob mice, wild-type mice and ob / ob mice. It was analyzed by comparing the biomechanical properties of long bones of type-ovx). To determine the breaking load, which is a measure of bone strength, a servo-hydraulic tester (at a constant displacement rate of 10 mm / min.
A three-point bend on Zurick GmbH & Co) was used to test the femur to fracture. The fracture load of bone from wild type and ob / ob mice was indistinguishable but significantly higher than that found in the bone of wild-type-ovx mice (Fig. ID). ). The results show that leptin deficiency has a beneficial effect on the biomechanical properties of bone. Bone analysis of db / db mice with sudden inactivation of the leptin receptor was also performed (Tarr).
aglia et al., 1995, Cell 83, 1263-1271). Like ob / ob mice, db / db mice are obese and have reduced sexual function. There was an increase in trabecular number in both long bones and spine similar to that observed in ob / ob mice (FIG. 1E), resulting in a 3-fold increase in bone volume compared to wild type mice (FIG. 1F). . This latter result genetically demonstrates that leptin signals through its known receptor to affect bone mass.

To determine whether the high bone mass of ob / ob mice could be explained by a phenotype by other endocrine abnormalities secondary to the absence of leptin signaling, the dysregulation of bone mass The levels of serum hormones that affect Ob / ob mice had normal levels of parathyroid hormone, whose deficiency causes high bone mass. The animals also had severe hypercortisolism, although IGFI and thyroxine levels were almost normal. This is extremely important because hypercortisol is the second most frequent cause of bone loss after gonadal failure as it inhibits bone formation. Thus, the high bone mass phenotype of ob / ob mice is
It occurs despite the coexistence of two conditions that favor bone loss: hypogonadism and hypercortisol.

6.3. High bone mass phenotype in ob / ob and db / db mice is secondary to absence of leptin signaling but not obesity High bone mass in ob / ob and ob / db mice The phenotype can be secondary to either lack of leptin signaling or obesity in these mice. To distinguish between these two possibilities, additional groups of mutant mice were analyzed for a high bone mass phenotype as in Example 2. First, ob / ob mice were fed a low-fat diet that postponed the appearance of obesity in ob / ob mice, and these mice had normal weight at 1 month of age. However, these mice already had a high bone mass phenotype at that age (Fig. 2A). Second, non-obese heterozygous leptin-deficient mice (ob / +) were analyzed. These animals also had a high bone mass phenotype (Figure 2B). Third, we observed bones from other mouse models of obesity that were not originally associated with leptin signaling. Another genetic model of obesity, Agouti yellow (A ^y / a) mice (Herberg and Coleman, 1977, Metabolism 26, 59-99), showed normal bone marrow like wild-type mice fed a high-fat diet. It had a quantity (Figs. 2C and 2D).

Thus, the presence of a high bone mass phenotype in ob / ob mice and the non-obese ob / + mice prior to the onset of obesity and its absence in a model of obesity independent of leptin is due to this high bone mass phenotype. It has been demonstrated that it is not obesity, but the absence of leptin signaling that is responsible for.

6.4. Increased osteoblastic function in ob / ob and db / db mice Increased bone mass results in increased osteoblastic bone formation, decreased osteoblastic bone resorption, or a combination of both abnormalities. It could have been due. To study osteoblastic function in vivo, as described (Duey et al., 1999, Genes Dev 13, 1025-
1036), and the rate of bone formation was quantified after double labeling with calcein, a newly formed bone marker (FIG. 3A). Calcein was injected twice at 8 day intervals and the animals were sacrificed 2 days later. In the 3 month and 6 month old ob / ob mice, 70% and 60% increase in bone formation rate was observed, respectively, compared to that of the wild type littermate (FIG. 3B). This result demonstrated that the high bone mass phenotype of ob / ob mice was at least in part due to increased osteogenic activity. Remarkably, considering the large increase in the rate of osteogenesis, the number of osteoblasts as well as the number of osteoblasts were not increased in ob / ob mice, and leptin deficiency contributed to the function of osteoblasts. It was shown to have an effect, but not its differentiation postnatally (FIG. 3C). Similarly, calcein labeling of db / db mice showed increased bone formation rates in both long bones and spine despite normal osteoblast numbers (
3D and 3E). The number of osteoclasts was increased in both mutant mouse strains as expected in view of the presence of hypogonadism (FIG. 3F), which
It suggests that the high bone mass phenotype of b / ob and db / db mice may have developed despite increased bone resorption.

The same group of control animals as described above was used to determine the specificity of the effect of absence of leptin signaling on osteoblast function. Both lean, 1-month-old ob / ob animals fed a low-fat diet and heterozygous leptin-deficient mice also significantly increased their bone formation rate (FIG. 3G). In contrast, two leptin-independent models of obesity, wild-type mice fed a high-fat diet and Ar / a
Mutant mice had normal bone formation parameters (Fig. 3H).

6.5. Analysis of osteoclast function in ob / ob mice The coexistence of a high bone mass phenotype and hypogonadism is so exceptional that it is deficient in bone resorption in these mice. Proposed a hypothesis. ob /
is a biochemical marker of bone resorption in ob mice (Eyre et al., 1988,
Biochem 252,494-500) Increased urinary clearance of deoxypyridinoline cross-links (Dpd) was a rebuttal to this hypothesis (wild type: 10.5 ± 2.5 nMDpd /
mM creatinine; ob / ob: 24.0b 4.0 nM Dpd / mM creatinine). The Pyrilinks-D-immunoassay kit (Metra Biosystem) was used to measure deoxypyridinoline crosslinks in urine upon waking. Creatinine values were used for inter-sample normalization (Creatininekit, Metra Biosystem). Circulating concentrations of 17β-estradiol and leptin were measured using the 3rd generation estradiol kit (Diagnostic
System Laboratories) and mouse leptin RIA kit (Linco) were used for quantification by radioimmunoassay. Estradiol is an estrogen analog and also a selective estrogen receptor modulator (SERM). Co-administration of an estrogen analog with an ob or obR inhibitor further increases the high bone mass phenotype.

Nevertheless, to address this more thoroughly, we utilized the hypogonadism of ob / ob mice. Hypogonadism usually leads to increased osteoclast numbers and bone resorption activity. Thus, if the osteoclasts of ob / ob mice were functional, correcting the hypogonadism in these mice would reduce their bone resorption rate by reducing the number of osteoclasts and thus their bone mass. Should be further increased. On the other hand, if the osteoclasts of ob / ob mice were not functioning properly, correcting their hyposexuality should not affect the severity of their high bone mass phenotype. To determine which of these two possibilities was correct, 17 β-estradiol or placebo pellets (Innovative Ressearch of America) were administered to 2 month old female ob /
Ob mice were subcutaneously transplanted to maintain a serum concentration of '250 pg / ml. These animals were analyzed after a treatment period of 3 months.

As expected, estradiol treatment corrected the hypogonadism, as judged by its uterine appearance and blood Enetradiol levels, and further resulted in normalization of osteoclast numbers. (FIG. 4C). It also led to a further increase in the high bone mass phenotype of these mice compared to placebo-treated ob / ob mice, thus eliminating defects in bone resorption as the cause of the high bone mass phenotype in ob / ob mice. (FIG. 4B). Ob / ob mice treated with estradiol showed a 50% increase in bone volume over wild-type mice treated with estradiol at the end of the treatment period, a 3-fold increase over untreated wild-type mice ( FIG. 4D).
Similar results were obtained in male ob / ob mice treated with testosterone grafts. Finally, the function of osteoclasts in ob / ob and db / db mice
It was studied in vitro in an assay using hematopoietic progenitor cells from wild-type, ob / ob or db / db mice and growth factors known to induce the differentiation of these cells into functional osteoclasts. Mouse osteoclasts were generated in vitro according to previously reported protocols (Simonet et al., 1997. Cell 89,3.
09-319; Quinn et al., 1998, Endocrinology 139, 4424-4.
427). Bone marrow cells of wild type, ob-ob and db / db mice were treated with 10% FB
S (Hiclone), mouse M-CSF 40ng / mL (Sigma), RANKL / ODF
10 6 on 24-well tissue culture plates or dentin chips in α-MEM (Sigma) containing 25 ng / mL (Peprotech), 10 ⁻⁸ M dexamethasone (Sigma) and 10 ⁻⁸ 1.25 dihydroxyvitamin D3. Cultured at an initial density of cells / well. The medium was changed every other day. After 6 days in culture, cells were fixed in 3.7% formalin. Osteoclasts formed on plastic plates were stained for tartrate-resistant acid phosphatase. Cells on dentin chips were removed by treatment with sodium hypochlorite. The dentin chips were then stained with toluidine blue to visualize the absorption pits.

As shown in FIG. 4E, wild-type, ob / ob and db / db hematopoietic progenitor cells differentiated equally well into osteoclasts and absorbed the matrix.

Thus, these experiments demonstrated that there were no detectable functional defects in osteoclasts in ob / ob and db / db mice, indicating high bone mass expression in these mouse mutant lines. It demonstrates that the pattern results exclusively from an increase in secondary bone formation associated with the absence of leptin signaling.

6.6. Absence of Leptin Signaling in Osteoblasts Previous analyzes have shown that leptin is an inhibitor of osteoblastic bone formation. Moreover, the presence of a high bone mass phenotype in db / db mice demonstrates that leptin must bind to its known receptor to perform this function. Theoretically, leptin is found in the fat directly to osteoblasts
It can act either indirectly through the release of the factor or by using the hypothalamic pathway as done for weight control. These three possible mechanisms of action were tested.

Levels of leptin expression in osteoblasts were maintained in 10% FBS mineralized medium for 4 days, then switched to 0.5% for 2 days and replaced daily to subconfluent levels from wild-type mice. Studied in primary osteoblast cultures. The medium was 80 ng / mL leptin (
Sigma) or 40 ng / mL Oncostatin M (R & D Diagnostics) or 20 in vehicle
It was replaced 2 hours before the treatment for 1 minute. RNA extraction was performed with triazole (Gibco). Northern blots were performed with 15 μg total RNA according to methods well known in the art.

Expression of leptin in osteoblasts was undetectable even after prolonged film exposure,
It showed that the probability of autocrine regulation was low (Fig. 5A). Leptin expression was also undetectable in whole bone specimens, providing an indirect rebuttal of paracrine regulation of osteoblastic function by leptin (FIG. 5A). In any case, functional leptin receptors must be present on osteoblasts for paracrine and / or endocrine regulation of osteoblast function by leptin. There are several transcripts of the leptin receptor, but only one, Ob / Rb, is believed to have signal transduction potential. (Tartaglia et al., 1995, Cell 83, 1263-1271; Chen
et al., 1996, Cell 84, 491-495; Lee et al., 1996, Nature.
379,632-635). The expression of this transcript of the leptin receptor is highly hypothalamic-specific, though not strictly. Ob-Rb in primary osteoblasts and whole bone preparation
RT-PCR experiments were performed to explore the transcripts. RT-P for Ob-Rb expression
CR analysis (27 cycles) was performed on the randomly primed cDNA with the following primers: 5'-TGGATAAACCCTTGCTCT.
TCA-3 ', and 5'-ACACTGTTAATTTTCACACCAGAG-
3 '(Friedman and Halaas, 1998, Nature 395, 763-770). Amplification of Hprt was used as an internal control of cDNA quality using the following primers: 5
'-GTTGAGAGATCATCTCCACC-3', and 5'-AGCGA
TGATGAACCAGGTTA-3 '. No Ob-Rb expression was detected in calvaria, long bones and primary osteoblast cultures in multiple experiments using the constant number of amplification cycles required to detect Ob-Rb transcripts in the hypothalamus. (FIG. 5B).

To determine if leptin could transduce its signal in osteoblasts, serum-starved primary osteoblasts isolated from wild-type mice were treated with leptin. Primary osteoblast cultures from calvaria of newborn wild type or db / db mice were established as previously described (Ducy et al., 1999, Genes Dev 13, 1025-103.
6), maintained in mineralized medium (αMEMI 0.1 mg / mL ascorbic acid: 5 mM α-glycerophosphate) supplemented with 10% PBS. Cultures of mutant cells were maintained in this medium for 15 days prior to analysis. Cultures derived from wild-type mice were maintained in this medium for 10 days, then the serum percentage was reduced to 0.5% and the medium was supplemented with 1.2 μg / mL leptin (Sigma) or vehicle for 5 days. did.

Expression of two immediate early genes whose transcription is increased after leptin treatment of target cells (Elmquist et al., 1997, Endocrinology 138, 839-842).
In addition, Stat, a downstream effector of leptin signaling in its target cells
Phosphorylation of 3 was monitored (Tartaglia et al., 1995, Cell 83, 1263-.
1271; Baumann et al., 1996, Proc Natl Acad Sci USA 93,83.
74-8378; Ghilardi et al., 1996, Proc Nati Acad Sci USA 93.
, 6231-6235; Vaisse et al., 1996, Nat Gebet 14, 95-9.
7). Oncostatin-M, which induces Stat3 phosphorylation and activates expression of the same two immediate early genes in osteoblasts, was used as a positive control (Levy et al., 1
996, Endocrinology 137, 1159-1165). Western blot analysis was performed to determine Stat3 phosphorylation as follows. Lyse the cells,
Protein extracts were separated on 7.5% SDS-PAGE and blotted on nitrocellulose (Biorad) for immunoblot assays by methods well known in the art. Follow the manufacturer's instructions for PhosphoPlus Stat3 (Tyr 7
05) Stat3 phosphorylation analysis was performed using the antibody kit (New England Biolabs).

As shown in FIG. 5C, treatment of osteoblasts with Oncostatin-M did induce Stat3 phosphorylation that did not occur with leptin treatment. Multiple physiological and supraphysiological doses of leptin were used in this experiment, but none of them were able to induce Stat3 phosphorylation. Similarly, Tis11 and c-fos expression, analyzed by standard Northern analysis, was not activated by leptin but rapidly and transiently by oncostatin-M (
FIG. 5D). Finally, the effect of long-term leptin treatment of primary osteoblast cultures on extracellular matrix synthesis and bone matrix mineralization was determined. The presence of collagen-rich extracellular matrix and mineralized nodules was assessed by whole mount staining of the cultures with Bungeson and von Kossa reagents, respectively. No differences were seen between control and leptin-treated cultures when assessing collagen synthesis or mineralized nodule formation.

Finally, in the absence of any functional leptin receptor on osteoblasts, wild type and db / db osteoblasts should be indistinguishable in ex vivo culture.
Primary cultures of osteoblasts from db / db and wild type mice were analyzed for their ability to produce and mineralize authentic bone extracellular matrix. There were no differences between wild-type and db / db primary osteoblast cultures for all analyzed parameters of alkaline phosphatase staining, type I collagen production and mineralized nodule formation (Fig. 5F). Taken together, these results indicate that the effect of leptin on bone formation in whole animals does not require binding of leptin to its receptor on osteoblasts.

6.7. In order to address the possibility that this effect of high bone mass leptin in the absence of adipose tissue could require the presence of fat, a dominant negative phenotype called A-ZIP is exclusively in adipocytes. We used a transgenic mouse model that expresses the above protein (Moitra et al. 1998, Genes Dev 12,3).
168-3181). This dominant negative protein is the DNA of most B-ZIP transcription factors, a class of transcription factors crucial for adipocyte differentiation.
Completely eliminate the binding ability. As a result, the A-ZIP / F1 transgenic mouse was
With no white adipose tissue, which is the fat type regulated by leptin signaling, the amount of inactive brown adipose tissue is dramatically reduced. These mice also have a 20-fold reduction in the leptin component (Moitra et al, 1998, Genes Dev 12,
3168-3181). In A-ZIP / FI transgenic mice, ob
A high bone mass phenotype was observed due to the same increase in osteoblastic function seen in the / ob and db / db mice (Figure 6).

This experiment has two implications. First of all, it confirms that leptin deficiency, but not high fat index, is responsible for the high bone mass phenotype in ob / ob and db / db mice. Second, it demonstrates that adipose tissue is not a relay element required for the action of leptin on bone formation.

6.8. Intracerebroventricular infusion of leptin corrects high bone mass phenotype in ob / ob mice Leptin binding to its hypothalamic receptor can rescue obesity in ob / ob mice, suggesting that leptin binding We addressed the question of whether or not the high bone mass phenotype could be corrected. For this purpose, a pump (8 ng / hr) delivering either PBS or leptin was inserted into the third ventricle of ob / ob mice as follows.
Animals were anesthetized with Avertin and placed on a stereotaxic apparatus (Stoelting). The calvaria was exposed and a 0.7 mm hole was drilled on the bregma. A 28-gauge cannula (Cerebral Infusion Kit U, Alza) was implanted in the third ventricle according to the coordinates midline, -0.3AP, 3mm compound side (0 point bregma). The cannula was fixed to the skull using cyanoacrylate and attached using a Tygon tube to an osmotic pump (Alza) placed in the dorsal subcutaneous space of the animal. The delivery rate was 0.25 ul / hr (leptin (Sigma) 8 ng / hr) PBS for 28 days. The dose has previously been shown to have no effect when administered systemically (Hallan et al., 1997, Proc Nati Acad Sci USA 94, 8878-8883). As close as possible to the biological status of the ob / ob mice, pumped animals were ovariectomized to avoid any artificial increase in bone mass due to hypogonadism correction.
The pumps were left in place for 28 days and double labeled with calcein to measure bone formation parameters.

Classical histology showed that the bones of leptin-treated mice regained their normal appearance, whereas those treated with PBS did not (FIG. 7A). They had relatively few trabeculae, and these trabeculae appeared to be more regular than in PBS-treated ob / ob mice. Bone volume, trabecular thickness, and rate of bone formation were all significantly reduced in leptin-treated mice (FIG. 7A). No leptin was detected in the sera of these animals using the specific radioimmunoassay. Bone phenotypic rescue by intraventricular infusion of leptin, coupled with the absence of measurable circulating leptin, indicates that the regulation of bone formation is a neuroendocrine leptin-dependent function.

6.9. Overexpression of soluble leptin receptor (ObRe) increases bone mass but does not result in obesity) Mice heterozygous for the absence of leptin (ob / +) have high bone mass It has a quantitative phenotype (Fig. 2B), suggesting that it may be possible to decouple the effect of leptin or leptin deficiency on bone mass from its effect on body weight. To test this, the apolipoprotein E promoter (ApoE-
Under the control of ObRe), transgenic mice overexpressing a cDNA encoding ObRe, a soluble form of the leptin receptor, were generated using standard techniques. The ApoE promoter is specifically expressed in the liver and is thus able to achieve high serum concentrations of any secreted protein to which it is bound.

The results of this experiment show that overexpression of ObRe in the serum of wild type mice does not result in obesity or infertility in both males and females at 3 months of age, but 50% to 100% of bone. It shows that it exactly does the result of the increase in the amount (Fig. 8).

This experiment, prior to and independent of its role on body weight, genetically confirms the role of leptin in the regulation of bone formation. This experiment also similarly confirmed that ObRe itself or any derivative of ObRe can be routinely tested or used as a bone formation enhancer for bone loss diseases such as osteoporosis. There is.

All patents and publications mentioned in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All patents and publications are given as if each individual publication was specifically and individually indicated to be included by reference.
Included as a reference in this book.

Those skilled in the art will readily appreciate that the patents of this invention are well adapted to carry out the ends and obtain the ends and advantages mentioned, as well as those inherent therein. You will recognize. Leptin, leptin receptor, leptin antibody, leptin analog, leptin antagonist, pharmaceutical composition described in this document,
Therapies, methods, procedures and techniques are presently representative of the preferred embodiments and were not intended to limit the scope of the subject matter, which was intended to be exemplary. One of ordinary skill in the art will recognize modifications and other uses for it that are encompassed within the spirit of the invention or defined by the scope of the appended claims.

[Brief description of drawings]

1A-1F show high bone mass phenotype in ob / ob and db / db mice. In other words, FIG. 1A shows an X-ray analysis chart of the vertebrae (vert) and long bones (femur) of 6-month-old wild type (wt) and ob / ob mice, and FIG. 1B shows 3-month-old (3 m) and 6-month-olds. Figure 6 shows histological analysis of bones of age (6m) (wt) and ob / ob mice. Two upper panels are spine analysis views and two lower panels are long bone analysis views. Petrified bone matrix is stained with von Kossa reagent. FIG. 1C shows a diagram quantifying the increase in bone volume in ob / ob mice. BV / TV represents bone volume relative to trabecular volume, gray bar indicates wt mouse, and black bar indicates ob / ob mouse. FIG. 1D shows wild type (wt), o
3 shows the results of a three-point bend analysis of the femurs of b / ob and wild type ovariectomized (wt-OVX) mice. FIG. 1E shows a histological analysis of the spines of 6 month old (wt) and db / db mice. FIG. 1F shows a diagram quantifying the increase in bone volume in db / db mice. The asterisk indicates a statistically significant difference between the two groups of mice (p <0.05). Error bars are standard mean deviation (SEM
) Is represented.

FIGS. 2A-2D show that the high bone mass phenotype in ob / ob mice is due to leptin deficiency and not obesity. That is, FIG. 2A
FIG. 3 shows a histological analysis of the spine of ob / ob mice (ob / ob 1 mo LF diet) fed a 1-month-old wt (wt1mo) and low-fat diet. FIG. 2B shows a histological analysis of the spine of 3 month old (wt) and ob / + mice. FIG. 2C shows a histological analysis of the spine of 6 month old (wt) and Aguti yellow mutant mice (A ^y / _a ). FIG. 2D shows a histological analysis of the spine of 6-month-old wt mice fed either a normal diet or a high fat (HF) diet. The underlined numbers indicate a statistically significant difference between the two experimental and control groups of mice (p <0.05).
).

FIG. 3 The disappearance of the leptin signal increases the function of hematopoietic cells. In FIG. 3A, calcei in 3 month old wild type (wt) and ob / ob mice.
n) Double labeling was demonstrated. The distance between the two labels (white arrows) represents the rate of bone formation. 3B-3F, ob / ob mice (B) and d
Bone formation rates in b / db mice (D) were increased by 45% and 70%, respectively, compared to littermates. This increase occurred in the presence of normal numbers of hematopoietic cells (FIGS. 3C and 3E) and, despite the increased number of hematopoietic cells due to hypogonadism (FIG. 3).
F), the rate of bone formation was increased compared to littermates. Here, white bars are wt mice; black bars are ob / ob mice; gray bars are db / db mice; 3 m.
Means a 3-month-old animal; 6 m. Means a 6-month-old animal, respectively. As shown in FIG. 4G,
Bone formation rates were also increased in fat-suppressed 1-month-old ob / ob and heterozygous ob / + mice (not obese) (weight shown in Figures 2A and B). As shown in FIG. 3H, overweight wt mice or A ^y / _a mice fed a high fat diet (see FIGS. 2C and D) were not leptin deficient but showed normal bone formation rates. Asterisk indicates statistically significant difference compared to control mouse (p <0.05). Error bars represent standard mean deviation (SEM).

4A-4E show that normal osteoclasts function in the absence of the leptin signal. Hypogonadism was corrected by treatment with 17β-estradiol (E2
) Or uncorrected (P, placebo) wild type (wt) and ob / ob mice are shown for comparative analysis. Figure 4A shows ob / o with 17β-estradiol treatment.
b Demonstrates that uterine degeneration in mice is corrected. As shown in FIGS. 4A-4D, as a result of histological analysis of the spine, a significant increase in trabecular bone was observed in 17β-estradiol-treated wt and 17β-estradiol-treated ob / ob mice by 17β-estradiol treatment. Gray bars represent wild-type mice; filled bars ob / ob mice; patterned bars therapeutic treatment mice; plain bars placebo control mice. Asterisks indicate statistically significant differences between treated and untreated mice (p <0.05). Error bars represent standard mean deviation (SEM). FIG. 4E shows e of ob / ob and db / db osteoclasts.
Normal differentiation and function in x-vivo has been demonstrated. wt, ob / ob and db
Bone marrow progenitors from the / db mouse are T
Differentiated equally well in RAP-positive (TRAP +) osteoclasts (upper panel and lower line).

5A-5F show the absence of signal from leptin in osteoblasts. Northern blot (No) of leptin expression in tissues and primary cells (non-petrified (NM) osteoblasts, mineralized (M) osteoblasts and chondrocytes) (upper panel).
The result of the rother blot) analysis is shown in FIG. 5A. Gapdh expression was used as an internal control for loading (lower panel). FIG. 5B shows O
It shows that b-Rb (Ob receptor, type b) transcription cannot be found in long bones, calvaria and primary osteoblasts by RT-PCR. This message is detected in the hypothalamus. Hprt as an internal control for cDNA quality
Was used. FIG. 5C is a Western Northern blot (Western
The result of the blot) analysis is shown. Oncostatin-M (OSM) was used as a positive control. FIG. 5D shows early gene expression (Tisll and cf) immediately after treatment of primary osteoblastic cultures with leptin or oncostatin-M.
os gene) Northern blot analysis results are shown. Gapdh expression was used as an internal control for loading (lower panel). FIG. 5E shows ex-v from wild-type mice maintained in the absence (vehicle) or presence of leptin.
Ivo primary osteoblast cultures are shown. Collagen synthesis (upper panel, van
Gieson staining or matrix petrification (lower panel, von Kossa)
No effect on the staining) was observed. FIG. 5F shows the normal function of db / db osteoblasts in the ex-vivo culture experiment. Collagen synthesis between primary osteoblastic cultures obtained from wt and db / db mice (upper panel, van
Gieson stain) or matrix mineralization (lower panel, von Kossa stain).

6A-6C show that adipose tissue is not required for development of the high bone mass phenotype. That is, FIG. 6A shows 6 months old wt and A-ZIP / without adipose tissue.
4 shows histological analysis of the spine of F-1 transcribed mice. Bone volume (B) and bone formation rate (C) in transcribed mice have been described. The asterisk is wt
And a statistically significant difference between the and transgenic mice (p <0.
05). Error bars represent standard mean deviation (SEM).

7A-7D show that the action of leptin on bone formation is mediated by the hypothalamic relay. That is, FIG. 7A shows that PBS or leptin is at the center (
FIG. 3 shows a histological comparison of the spines of a 4 month old ob / ob mouse and a wt mouse, which were infused in the third bench cool). Experiments were performed on bone volume (B), trabecular bone volume (C) and bone formation rate (D). The asterisk indicates a statistically significant difference between mice infused with PBS and mice infused with leptin (p <0.0.
5). Error bars represent standard mean deviation (SEM).

FIG. 8. Soluble leptin receptor increases bone mass without causing increased obesity.
In FIG. 8, male and female transcribed gene mice (containing the ApoE-ObRe transcribed gene) were bred. At 3 months of age, these mice were compared to wild type mice for bone volume / total volume and body weight. There was no significant increase in body weight during the 3 months, but an increase in bone mass of 50% to 100% was observed. It should be noted that the drawings and the numerical values do not necessarily correspond to the scale, and for easy understanding, some parts are exaggerated and schematically shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 38/43 A61K 39/395 P 4C206 39/395 48/00 A61P 19/08 48/00 19/10 A61P 19/08 43/00 111 19/10 C12Q 1/02 ZNA 43/00 111 G01N 33/15 Z C12Q 1/02 ZNA 33/50 Z G01N 33/15 33/566 33/50 A61K 37/48 33/566 37/02 (31) Priority claim number 09 / 489,873 (32) Priority date January 20, 2000 (January 20, 2000) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP (72) Invention By Duchy, Patricia United States 7 7098 Apartment 280, Hughston, TX, Apartment 280, Revelle Street 2701 (72) Inventor Amring, Michael Germany Dee 22607 Hamburg, Grossflot Becker Strasse 49 Beef Term (reference) 2G045 AA40 CB13 FB03 4B063 QA01 QA20 QQ08 4C084 AA02 AA13 AA17 AA19 AA20 BA44 DA39 DB63 DC01 MA02 NA14 ZA962 ZA972 ZC112 4C085 AA13 AA14 BB07 DD21 DD62 DD63 EE01 4C086 MA02 ZA04 A02 A04 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A04 A01 A02 A01 A02 A04 A01 A02 A11 A02 A02 ZA97 ZC42

Claims (60)

[Claims] 1. A method of treating a bone disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that reduces serum leptin levels, the bone disease corresponding thereto. A method which is characterized by a decrease in bone mass relative to that of non-diseased bone. 2. The method according to claim 1, wherein the leptin level is decreased due to a decrease in leptin synthesis. 3. The method according to claim 2, wherein the compound is a triple helix sequence of a polynucleotide encoding antisense, ribozyme or leptin. 4. The method according to claim 1, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, and osteoarthritis. 5. A method of treating a bone disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that reduces leptin levels in cerebrospinal fluid, the bone disease being addressed. A method characterized by a decrease in bone mass relative to that of non-diseased bone. 6. The compound according to claim 5, which binds to leptin in blood.
The method described. 7. The method according to claim 6, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, and osteoporosis. 8. A method of treating a bone disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound, the method comprising comparing the bone disease to a corresponding non-diseased bone. Characterized by a decrease in bone mass, the compound consisting of an antibody that specifically binds leptin, a soluble leptin receptor polypeptide, an inter-alpha-trypsin-inhibitor heavy chain-related protein, and an alpha2-macroglobulin protein How to choose from. 9. The method according to claim 8, wherein the antibody is a monoclonal antibody. 10. The method according to claim 8, wherein the antibody is a human or chimeric antibody. 11. The method according to claim 10, wherein the antibody is a humanized antibody. 12. The method according to claim 8, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, and osteoporosis. 13. A method of treating a bone disease comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that reduces the level of phosphorylated Stat3 polypeptide, said bone disease comprising: A method which is characterized by a decrease in bone mass relative to that of the corresponding non-diseased bone. 14. The compound according to claim 1, which is a leptin receptor antagonist.
3. The method described in 3. 15. The method of claim 14, wherein the leptin receptor antagonist is acetylphenol. 16. The method according to claim 14, wherein the leptin receptor antagonist is selected from the group consisting of an antibody that specifically binds to leptin and an antibody that specifically binds to the leptin receptor. 17. The method according to claim 13, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, and osteoporosis. 18. The method of claim 1, 5 or 13 further comprising administering to the mammal a therapeutically effective amount of a selective estrogen receptor modulator. 19. The method of claim 18, wherein the selective estrogen receptor modulator is estradiol. 20. A method for preventing bone disease, which comprises administering a compound that lowers leptin level in serum to a mammal at risk of bone disease at a concentration sufficient to prevent bone disease. , The bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. 21. The method according to claim 20, wherein the leptin level is decreased due to a decrease in leptin synthesis. 22. The method of claim 21, wherein said compound is a triple helix sequence of a polynucleotide encoding an antisense, ribozyme or leptin. 23. The method according to claim 20, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, and osteoporosis. 24. A method for preventing bone disease, which comprises administering a compound that reduces the level of leptin in cerebrospinal fluid to a mammal at risk of bone disease at a concentration sufficient to prevent bone disease. And the bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. 25. The method of claim 24, wherein said compound binds leptin in blood. 26. The method according to claim 24, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia and osteoarthritis. 27. A method of preventing bone disease comprising administering to a mammal at risk of bone disease the compound at a concentration sufficient to prevent bone disease, the non-bone disease corresponding to Characterized by a decrease in bone mass relative to that of diseased bone, the compound comprising an antibody that specifically binds leptin, a soluble leptin receptor polypeptide,
A method selected from the group consisting of inter-alpha-trypsin-inhibitor heavy chain-related protein, and alpha2-macroglobulin protein. 28. The method of claim 27, wherein the antibody is a monoclonal antibody. 29. The method of claim 27, wherein said antibody is a human or chimeric antibody. 30. The method of claim 29, wherein said antibody is a humanized antibody. 31. The method according to claim 27, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia and osteoarthritis. 32. A method of preventing bone disease comprising administering to a mammal at risk of bone disease a compound that reduces the level of phosphorylated Stat3 polypeptide in a concentration sufficient to prevent bone disease. And wherein said bone disease is characterized by a decrease in bone mass as compared to that of the corresponding non-diseased bone. 33. The compound according to claim 3, which is a leptin receptor antagonist.
2. The method described in 2. 34. The method of claim 33, wherein the leptin receptor antagonist is acetylphenol. 35. The method according to claim 33, wherein the leptin receptor antagonist is selected from the group consisting of an antibody that specifically binds to leptin and an antibody that specifically binds to the leptin receptor. 36. The method according to claim 32, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia and osteoarthritis. 37. A method for diagnosing a bone disease in a mammal, comprising: (a) measuring the leptin level in the serum of the mammal; and (b) the level measured in (a) above in the control serum. If the level obtained in (a) above is higher than that of the control, the mammal is diagnosed as having a bone disease and the bone disease is A method which is characterized by a decrease in bone mass relative to that of diseased bone. 38. The method of claim 37, wherein the mammal is a human. 39. The method according to claim 37, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia and osteoarthritis. 40. A method for diagnosing a bone disease in a mammal, comprising: (a) measuring the leptin level in the cerebrospinal fluid of the mammal; (b) using the level measured in (a) above as a control marrow. If the level obtained in (a) above is higher than that of the control, including comparing with leptin level in the liquid, the mammal is diagnosed as having a bone disease and said bone disease is A method characterized by a decrease in bone mass relative to that of non-diseased bone. 41. The method of claim 40, wherein said mammal is a human. 42. The method according to claim 40, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia and osteoarthritis. 43. A method for determining whether a test compound has the ability to regulate bone mass in a mammal, comprising: (a) contacting the test compound with a polypeptide, and (b) using the test compound as the polypeptide. Measuring whether or not to bind,
When the test compound binds to the polypeptide, it is confirmed that the test compound has the ability to regulate bone mass, and the polypeptide is selected from the group consisting of leptin polypeptide and leptin receptor / polypeptide. How to be one. 44. The method of claim 43, wherein said polypeptide is a human polypeptide. 45. The ability to regulate bone mass is the ability to increase bone mass.
3. The method described in 3. 46. The ability to regulate bone mass is the ability to reduce bone mass.
3. The method described in 3. 47. A method for identifying a compound that regulates bone mass in a mammal, comprising: (a) contacting a test compound with a polypeptide; and (b) identifying the test compound bound to the polypeptide, And (c) the test compound of (b) above is administered to a non-human mammal, and the test compound determines the bone mass of the mammal corresponding to the bone of a non-treated control non-human mammal. Determining whether it modulates relative to the amount, wherein the polypeptide is selected from the group consisting of leptin polypeptides and leptin receptors / polypeptides, wherein the compound If modulating, a method wherein a compound that modulates bone mass in a mammal is identified. 48. The method of claim 47, wherein said polypeptide is a human polypeptide. 49. The ability to regulate bone mass is the ability to increase bone mass.
7. The method according to 7. 50. The ability to regulate bone mass is the ability to reduce bone mass.
7. The method according to 7. 51. A method for confirming whether a test compound has the ability to regulate bone mass in a mammal, comprising: (a) using a test compound as a leptin polypeptide and a leptin receptor / polypeptide, and a leptin / leptin receptor. Contacting for a time sufficient for the body complex to form and (b) measuring leptin / leptin receptor complex levels, the measured level being measured in the absence of the test compound. The test compound is confirmed to have the ability to regulate bone mass when the level is different from the above-mentioned level. 52. The method of claim 51, wherein the leptin polypeptide is a human polypeptide. 53. The method according to claim 51, wherein the leptin receptor / polypeptide is a human polypeptide. 54. The ability to regulate bone mass is the ability to increase bone mass.
The method described in 1. 55. The ability to regulate bone mass is the ability to reduce bone mass.
The method described in 1. 56. A method of determining whether a test compound has the ability to reduce bone mass in a mammal, comprising: (a) contacting the test compound with cells expressing a functional leptin receptor, and (b) ) Measuring whether the test compound activates the leptin receptor, and when the test compound activates the leptin receptor, the test compound has the ability to reduce bone mass in a mammal. How to be confirmed. 57. A method for identifying a compound that reduces bone mass in a mammal, comprising: (a) contacting a test compound with a cell expressing a functional leptin receptor, the test compound activating the leptin receptor. Whether or not the test compound is confirmed to be activated, and (b) the test compound confirmed to activate the leptin receptor in (a) above is administered to a non-human animal, and the test compound controls the bone mass of the animal. Determining whether or not the bone mass of a corresponding bone of a non-human animal is decreased, and when the test compound reduces bone mass, a compound that reduces bone mass in a mammal is identified How to be. 58. A method for confirming whether a test compound has the ability to increase bone mass in a mammal, comprising: (a) contacting a leptin polypeptide and a test compound with a cell expressing a functional leptin receptor. And (b) determining whether the test compound reduces activation of the leptin receptor as compared to that observed in the absence of the test compound, the activation of the leptin receptor being A method wherein a test compound to be reduced is confirmed to have the ability to increase bone mass in a mammal. 59. A method for identifying a compound that increases bone mass in a mammal, comprising: (a) contacting a leptin polypeptide and a test compound with a cell expressing a functional leptin receptor, wherein the test compound is leptin. It is determined whether or not the activation of the receptor is decreased, and (b) the test compound confirmed to reduce the activation of the leptin receptor in (a) above is administered to a non-human animal to obtain the test compound. Determines whether the bone mass of the animal is increased compared to the bone mass of the corresponding bone of a control non-human animal, and if the test compound increases the bone mass of the animal, the bone mass in the mammal is The method wherein the compound that augments is identified. 60. The activation of the leptin receptor is measured by measuring the level of phosphorylated Stat3 polypeptide, 56, 57, 5
The method according to any of 8, 59.