WO2008094875A2 - Methods for treating bone loss and bone disease associated with chronic kidney disease - Google Patents

Abstract

Disclosed are methods and preparations for the treatment and or inhibition of bone loss in uremic animals. Leptin and melanocortin (particularly melanocortin 4 receptor or melanocortin 3 receptor) signaling systems are employed as part of an overall treatment regimen for such animals. Methods for reducing bone fracturability, preserving bone mineral density, maintaining tensile bone strength, and promoting and preserving bone health in patients having renal osteodystrophy, including chronic renal disease (CKD) and end-stage renal disease (ESRD), are also provided. A screening method for selecting candidate compounds that inhibit melanocortin receptor activity is also provided, wherein selected candidate compounds may be used in the formulation of a pharmaceutical preparation for the treatment of uremic patients having or at risk of developing compromised bone health, such as osteodystrophy.

Description

METHODS FOR TREATING BONE LOSS AND BONE DISEASE ASSOCIATED WITH CHRONIC KIDNEY DISEASE

STATEMENT OF FEDERAL GOVERNMENTAL SUPPORT

[0001] The United States Government may have rights to the invention described herein as research relevant to the development of the invention was funded by United States governmental grant funds NIH/NIDDK K24DK059475.

CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the priority of U.S. Provisional No. 60/897,869, entitled "Methods for Treating Bone Loss and Bone Disease Associated with Chronic Kidney Disease," filed January 29, 2007, the entire disclosure and contents of which is hereby incorporated by reference.

BACKGROUND Field of the Invention

[0003] The present invention relates generally to the field of pharmacologically active preparations and particularly to the selection (screening) and preparation of pharmacologically active preparations that are useful in the treatment and/or prevention of osteodystrophy associated with chronic kidney disease (CKD), uremia and end-stage renal disease (ESRD). The present invention also relates to the field of renal osteodystrophy, renal bone disease, uremic osteodystrophy and uremic bone disease, as methods are also provided for the treatment of these conditions.

Related Art

[0004] Bone disease can result in considerable morbidity for patients with CKD and ESRD. Reductions in bone mineral density (BMD) are common in ESRD patients and may result in an increased susceptibility to low-trauma fracture (23, 24). In one study of chronic hemodialysis patients, 48.9% had reduced BMD; and in 19.3%, BMD was below the fracture threshold (23) as defined by the World Health Organization (21). The incidence of spontaneous skeletal fractures has been shown to be three to four times higher in dialysis patients than in the normal population (24). Neither the use of biochemical markers, nor the use of bone biopsy, can predict either the rate of bone loss or the risk of skeletal fractures in these patients. The etiology of this reduced bone mineral density and increased fracture risk in uremic bone disease is not well understood. Although hyperparathyroidism is a major factor in uremic bone disease, several studies have been unable to demonstrate an association between reduced bone density and parathyroid hormone levels (25), suggesting other factors may be involved. [0005] Recent evidence suggests that leptin, in addition to its primary function to regulate energy expenditure and fat mass, also plays a key role in skeletal physiology. Leptin is a small polypeptide hormone secreted primarily by adipocytes. Leptin-deficient (ob/ob), leptin receptor-deficient (db/db) and lipodystrophic mice, all of which exhibit decreased leptin signaling, have the same high bone mass (HBM) phenotype (1). That these three mutant mouse strains, characterized by a great disparity in body weight, display the same HBM phenotype, demonstrates that it is leptin signaling, not body weight, which is influencing bone mass. Histomorphometric and physiologic analyses demonstrate that leptin is a selective inhibitor of bone formation. There is no evidence of leptin signaling in osteoblasts. Fat is not required for the development of the HBM phenotype, but intracerebroventricular infusion of leptin completely reverses the HBM phenotype of the ob/ob mice and induces bone loss in wild-type (WT) mice (1). Thus leptin is a potent inhibitor of bone formation acting through the central nervous system.

[0006] Leptin is cleared from the circulation by the kidney by glomerular filtration followed by metabolic degradation in the renal tubules (3, 5). In patients with normal renal function, there is a net renal intake of 12% of circulating leptin, whereas in patients with ESRD, there is no renal update of leptin. In hemodialysis patients, serum leptin levels (factored for body mass index) were increased four- fold compared to healthy controls. Serum leptin is not effectively cleared by hemodialysis with cellulose membranes (5). In a cross- sectional study with various degrees of chronic kidney diseases, significant correlations were found between leptin levels and glomerular filtration rate and body mass index (4). High circulating leptin concentrations are reported in diseases typically associated with osteopenia, such as liver cirrhosis, type 2 diabetes (22) and ESRD (6). An inverse correlation between serum leptin levels and histomorphometric indicators of bone turnover has been demonstrated in renal bone disease associated with ESRD. Serum leptin inversely correlated with parathyroid hormone, bone formation rate and mineral apposition rate in chronic dialysis patients. A complementary analysis in the same study in male dialysis patients revealed that the risk for low bone turnover increases in parallel with serum leptin concentrations, being 5 times higher in patients with high serum leptin (third tertile) than those with relatively low serum leptin (first tertile) (23).

[0007] Serum leptin level is the main, if not the only, means by which leptin regulates bone mass. Leptin's antiosteogenic and anorexigenic functions are mediated by similar concentrations of leptin. Increasing leptin levels dramatically reduces bone mass. Conversely, reducing serum-free leptin level increases bone mass. The high bone mass of lipodystrophic mice can be corrected by restoring normal serum leptin levels, suggesting that leptin is an adipokine both necessary and sufficient to control bone mass (2). Leptin signals centrally through hypothalamic pro-opio-melanocortin (POMC) neurons and melanocortin receptors affecting appetite and metabolic rate. POMC neurons in the hypothalamus and brainstem regulate body weight via their release of α-MSH that in turn activates the melanocortin 4 receptor (MC4-R) in many parts of the brain causing a decrease in appetite and increase in metabolic rate. Melanocortin 3 receptor (MC3-R) is thought to function as an inhibitory autoreceptor on POMC neurons. Peripheral injections of MC3-R agonists stimulate feeding both in wild-type mice as well as in MC4-RK0 mice indicating that this activity is independent of signaling through the MC4-R. MC3-R may also inhibit POMC neuronal activity through an increase in the activation of GABAergic neuropeptide Y neurons in the arcuate nucleus (39).

[0008] MC4-R knockout mice display increased bone mass and strength (26). Patients with loss-of- function mutations of MC4-R have markedly increased total body bone mineral content and increased bone mineral density, out of proportion to their obesity (28), suggesting that leptin may regulate bone mass by signaling through this receptor. The present investigators have recently shown that leptin's hypothalamic melanocortin pathway plays a significant role in the pathogenesis of uremic cachexia (11). The role of leptin signaling through the MC4-R in uremic bone disease has not been examined.

[0009] Peripheral mediators of leptin's antiosteogenic function appear to be neuronal. Leptin deficiency results in low sympathetic tone and genetic or pharmacological ablation of adrenergic signaling leads to a leptin-resistant state of high bone mass, β-adrenergic receptors on osteoblasts regulate their proliferation, and a β-adrenergic agonist decreases bone mass in leptin-deficient and wild-type mice while a β-adrenergic antagonist increases bone mass in wild-type and ovariectomized mice (34,35). Sympathetic signaling, via β-adrenergic receptors present on osteoblasts, may serve to control bone formation downstream of leptin. That bone resorption cannot increase in gonadectomized β-adrenergic receptor-deficient mice highlights the biological importance of this regulation, but also contrasts sharply with the increase in bone resorption characterizing another hypogonadal mouse with low sympathetic tone, the ob/ob mouse. This discrepancy is explained, in part, by the fact that cocaine amphetamine-regulated transcript (CART), a neuropeptide whose expression is controlled by leptin and nearly abolished in ob/ob mice, inhibits bone resorption (34). Thus, leptin- regulated neural pathways control both bone formation by osteoblasts and bone resorption by osteoclasts. Another series of studies was conducted to determine if the neuronal signaling was through the sympathetic nervous system rather than the adrenergic hormones in mediating effects on bone mass regulation. Mutant mice deficient in dopamine β-hydroxylase (Dbh), an enzyme necessary to produce catecholamine ligands (norepinephrine and epinephrine) for the adrenergic receptor, also have high bone mass (HBM). This finding is significant because Dbh^7" mice have an increase in serum corticosterone and dopamine levels, two conditions favoring low bone mass. The HBM in Dbh^{" "} mice is not associated with hyperinsulinism or any other hormonal perturbation, except for corticosterone and dopamine (35).

[0010] Catecholamines are released from two main sources: the sympathetic nerves and the adrenal glands. To determine whether the adrenal production of catecholamines, the main source of circulating epinephrine, was responsible for the skeletal phenotype, Takeda and colleagues analyzed WT mice in which the adrenal medulla had been surgically removed. Histological analysis showed that the removal of the adrenal medulla did not affect bone mass. These results establish the existence of a neuronal regulation of bone formation by the sympathetic nervous system (35).

[0011] A need continues to exist in the medical arts for effective methods in the treatment and/or prevention of bone loss and bone disease, particularly bone loss and bone disease attendant CKD and ESRD in an afflicted person, and the development of novel therapeutic compositions and strategies for treatment of these debilitating complications of renal disease.

SUMMARY

[0012] In a general sense, the present invention provides methods for reducing, treating and/or inhibiting bone loss or osteodystrophy attendant renal disease, particularly CKD and

ESRD, in a mammal, particularly in a human. One aspect of the invention comprises treating a human having or at risk of developing bone loss or osteodystrophy that is associated with renal disease with a therapeutically effective amount of a substance capable of blocking leptin or melanocortin signaling, thus reducing the bone loss or osteodystrophy associated with the renal disease.

[0013] In some embodiments, the reduction of bone loss or osteodystrophy in individuals experiencing renal disease resulting from treatment of the individual with the substance may be accompanied by reduced or maintained bone fracturability, an increase or maintenance in bone mineral density (BMD), or maintained or increased tensile bone strength relative to individuals experiencing renal disease that did not receive the substance.

[0014] The substance capable of blocking leptin or melanocortin receptor signaling may be further described as an inhibitor of a melanocortin receptor, such as a melanocortin receptor antagonist or reverse agonist. In some embodiments, the melanocortin receptor inhibitor is a melanocortin-4 receptor (MC4-R) antagonist. The MC4-R inhibitor may be further described as being essentially free of melanocortin-3 receptor (MC3-R) antagonistic activity. [0015] The MC4-R antagonists or reverse agonists useful in the described preparations and methods may be further described as having a Ki value of about 1.5 to about 2.0 nM. In particular embodiments, the melanocortin-4 receptor antagonist may be described as having a Ki of at least about 1.8 nM. By way of example, such MC4-R antagonists include: agouti- related protein or peptide (AgRP), 4-{(2R)-[3-Aminopropionylamido]-3-(2,4-dichlorophenyl) propionyl}-l-{2-[2-thienyl)ethylaminomethyl]phenyl}piperazine; SHU9119; HS014;

Corticotropin-releasing hormone (CRH) antagonist; or alpha helical-CRH(9-41).

[0016] In another aspect, the substance may be described as a melanocortin-3 receptor (MC3-R) specific agonist. In some embodiments, the MC3-R specific agonist is D-Trp⁸-γ- MSH. The MC3-R agonist may be further described as being essentially free of MC4-R antagonistic activity.

[0017] In other embodiments, the substance may be described as a POMC neuron inhibitor or an activator of NPY neurons, such as NPY.

[0018] The present invention provides for a screening method for identifying a candidate compound for use in the treatment of bone loss and osteodystrophy in a mammal experiencing renal disease is provided. In some embodiments, the method comprises exposing a control culture of cells expressing an MC4 receptor to an agonist such as melanocyte stimulating hormone (MSH) or melanotan (MT) II to provide an activated control measure of melanocortin 4 receptor activity. The activated control test culture of cells is then exposed to a known MC4 receptor antagonist or reverse agonist, such as agouti-related peptide (AgRP) to provide a measure of control MC4-R inhibitory activity. A second test culture of activated cells expressing the MC4-R receptor is then exposed to a candidate substance together with the MC4-R agonist, and a measure of the inhibitory activity is determined to provide a candidate substance test measure of MC4-R inhibitory activity. A candidate substance may be selected that is determined to have an MC4-R inhibitory activity that provides at least 50% or more of the MC4-R inhibitory activity observed in the control culture with AgRP following the MC4-R agonist. [0019] The present invention provides an alternative screening method for identifying a candidate substance for use in the treatment of bone loss attendant renal disease in a mammal. This screening method comprises expressing a melanocortin-3 receptor in a culture of cells. The cells expressing the MC3 receptor are exposed to an MC-3 receptor agonist, such as D- Trp-γ-MSH, to provide a control measure of activated cell MC3-R activity. Separately, a second test culture of activated cells expressing the MC3-R is exposed to a candidate substance. A candidate substance is selected if it demonstrates 50% or more of the control substance MC3-R agonist activity. In some embodiments, the selected candidate substance is essentially free (for example, 10% or less) of melanocortin 4-receptor agonist activity.

[0020] In some embodiments of the screening method, the cells are a human cell line, such as HER 293 cells, that express MC4 receptor, MC3 receptor, or both. In some embodiments, an acceptable level of melanocortin receptor inhibitory activity is about from 50% to about 85%, or about 50% to about 75%, or about 60% or more inhibition of melanocortin receptor activity as compared to control culture melanocortin receptor inhibitory activity, under similar conditions. [0021] As used in the description of the present method, melanocortin receptor activity may be measured by the level of cyclic AMP activity. According to this embodiment, after cells are exposed to a candidate substance for a predetermined length of time, the cells are lysed and the level of cAMP is measured. The level of cAMP activity may be quantified using the TropixcAMP-Screen lumiluminescent enzyme-linked immunosorbent assay system (Applied Biosystems) according to the manufacturer's instructions. Therefore, a candidate substance comprising a MC4-R antagonist or reverse agonist or a MC3-R agonist would be selected from the screen on the basis of a reduced level of cAMP. Alternatively, a candidate MC4-R antagonist may be selected on the basis of its ability to compete with radiolabeled NDP-MSH for binding to the MC4 receptor.

[0022] In some embodiments of the above screening methods, a further step may be included wherein a selected candidate substance demonstrating an acceptable level of cAMP inhibitory activity or competition with NDP-MSH binding to MC4-R may be subjected to an in vivo screening step. In such embodiments, the in vivo screening step will comprise examining the activity of the selected candidate substance in an in vivo model for uremic bone disease. Accordingly, a candidate substance will be further selected if it manifests an inhibition of bone loss in this in vivo model relative to bone loss observed in a diseased (uremic) animal that did not receive the candidate substance. In some embodiments, this second step of in vivo screening will be conducted in a uremic mouse model described herein.

[0023] In yet another aspect, the invention provides a pharmaceutical composition comprising a substance possessing melanocortin-4 receptor inhibitor activity, such as an MC4 receptor antagonist or reverse agonist. In yet another aspect, the invention provides a pharmaceutical composition comprising a substance possessing melanocortin-3 receptor agonist activity. These pharmaceutical compositions may further include a pharmaceutically acceptable carrier.

[0024] The following abbreviations are used throughout the description of the present invention:

[0025] AgRP Agouti-related Protein;

[0026] POMC Pro-opiomelanocortin;

[0027] MC4-R Melanocortin-4 receptor;

[0028] MC3-R Melanocortin-3 receptor;

[0029] NPY Neuropeptide Y;

[0030] α-MSH alpha melanocyte stimulating hormone;

[0031] BMD Bone mineral density;

[0032] HBM High bone mass;

[0033] icv Intracerebroventricular cannulation;

[0034] WT Wild-type. Brief Description of the Drawings

[0035] The invention will be described in conjunction with the accompanying drawings, in which: [0036] Figures IA- IE, according to some aspects of the invention, presents an in vivo study wherein mice were weighed to the nearest 0.1 g (IA) and then anesthetized for in vivo scanning by dual-energy X-ray absorptiometry (DEXA) to determine whole body bone mineral content (BMC) (IB), bone mineral density (BMD) (1C), lean mass (ID), and fat mass (IE). Initial (0) and final (6) values from 6-week study are presented as well as the change (%). Data are expressed as means ± SE. * p<0.0001

[0037] Figures 2A - 2E, according to some aspects of the invention, presents a measure of bone mineral content (BMC) and bone mineral density (BMD) of the isolated femora assessed by dual energy X-ray absorptiometry (2A and 2B, respectively). Geometric measures of the cortical area (2C) and cortical thickness (2D) of the femoral mid-shaft diaphysis were determined by micro μCT scanning. Femora were further tested for failure in response to three-point bending. The ultimate force (load) necessary for femoral shaft failure was expressed in Newtons (N) (2E). Data are expressed as means ± SE. * pO.0001.

[0038] Figure 3, according to some aspects of the invention, presents a 14-day study wherein WT-N/AgRP and WT-N mice were fed ad libitum, whereas WT-S mice were pair- fed with WT-N/AgRP mice (S = sham; N = nephrectomized). Serum leptin levels are calculated as ng/mL. Data are expressed as means ± SE.

[0039] Figure 4, according to some aspects of the invention, shows three-dimensional architecture of the midshaft femur bone area assessed by μCT scanning for WT-S, WT-N, WT-N/V, and WT-N/AgRP mice.

Detailed Description of the Preferred Embodiments

[0040] It is advantageous to define several terms before describing the invention. It should be appreciated that the following definitions are used throughout this application.

Definitions [0041] Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.

[0042] The term "a", "an" and "the" include reference to the plural unless the context as herein represented clearly indicates otherwise.

[0043] The term "biologically compatible form suitable for administration in vivo" relates to a bioavailable compound or derivative thereof that when administered peripherally, i.e. orally, intravenously, intramuscularly or intraperitoneally, that it has good CNS penetration to exert its therapeutic effects on bone disease through leptin or melanocortin signaling with toxic side effects being outweighed by the therapeutic benefits. The compounds and preparations containing the compounds may be administered to living organisms, including humans, animals, and plants.

[0044] The term "bone loss" relates to a reduction in bone mass (determined as a measure of bone mineral density of a single femoral bone determined by dual energy X-ray absorptiometry (DEXA)), or a reduction in bone mass measured for an entire animal (determined with a pencil beam densiometer), or a decrease in dry bone weight in grams (determined as an extrapolated value determined from the lean (muscle) mass), or a decrease in bone fracturability (determined as a measure of biomechanical strength determined by a 3- point bending apparatus with a high resolution material testing apparatus), or a decrese in tensile bone strength, or alteration of geometry and microarchitecture of femoral specimens (determined by a X-ray microtomographic scanner). [0045] The term "therapeutically effective amount" relates to an amount effective to achieve a desired result. The therapeutically effective amount may, for example, vary according to factors such as the disease state, age, sex, and weight of an organism, animal or individual, and the relative potency of the compound to elicit a desired response in or on an organism, animal or human individual. A dosage regimen may be adjusted to provide an optimum therapeutic response. For example, several different doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. [0046] The term "antagonist" as used in the description of the present invention refers to a compound that has an inhibitory effect on a receptor, particularly a melanocortin receptor, through competitive binding to the receptor in place of another ligand or agonist. The term "reverse agonist" as used in the description of the present invention refers to a compound that has an inhibitory effect on a receptor, particularly a melanocortin receptor, by a mechanism other than competitive binding, such as by triggering an inverse or reverse effect on the receptor compared to an agonist. The term "agonist" is a compound that binds to a specific receptor and triggers a response, typically a positive response, in the cell.

[0047] A "mammal" as used in this specification refers to any mammalian species that may potentially experience osteodystrophy or bone loss associated with renal disease. The term "mammal" is intended to include humans, monkeys, and other primates. The term

"mammal" further refers to other animals having an agricultural purpose or domesticated use including, but not limited to, cattle, sheep, goats, pigs, chickens, roosters, horses, canines, cats, etc. The term "mammal" also refers to animals having research or laboratory uses including, but not limited to, rabbits, mice, rats, etc.

Description

[0048] While the present invention is not necessarily limited to such applications, various aspects of the invention may be appreciated through a discussion of the several examples presented below using this context.

Example 1 - Material and Methods

[0049] The present example provides a description of the materials and methods used in the practice and description of the present invention. All methodologies have been established in the present investigators laboratories and have been published (11, 15). Therefore, only brief descriptions will be provided.

Animals

[0050] Wild-type (WT) mice were obtained from the Jackson laboratory. C57BL/6J

MC4-R KO mice were obtained from the Roger Cone laboratory and continuously bred in the inventor's laboratory. MC4-R KO mice were derived from the original C57BL/6Jxl29 colony and were backcrossed 10 times to C57BL/6J strain. Homozygous MC4-R KO mice are viable and fertile. All mice are raised group-housed in a 12 h light, 12 h dark cycles. For studies measuring food intake, mice are housed individually and food intake estimated by measuring the weight of powdered food remaining in feeding chambers designed to maximize spill capture. All mice are weaned at 21 days, and mice are either fed ad libitum or pair- fed with powdered Laboratory Rodent Diet 5015 containing 17% crude protein (LabDiet, Richmond, IN) that is weighed and replaced daily. Male animals, aged 8-10 weeks, are used. All studies were conducted according to the NIH Guide for the Care and Use of Laboratory Animals.

Standard subtotal nephrectomy and sham operation

[0051] Uremia is induced in the animals by standard subtotal nephrectomy operation, in a two-stage procedure as described (11). For each successfully nephrectomized animal, another sham-control operation is subsequently performed in a control animal.

Intracerebroventricular (lev) cannulation

[0052] The detailed procedure is as described previously (11). Briefly, mice are anesthetized. A small midline incision over the dorsal scalp is made to allow access to the cranium. A sterile guide cannula with obturator stylet (Small Parts, Inc., Miami Lakes, FL) is stereotaxically implanted in the lateral ventricle. The cannula is then fixed in place with dental cement (Yates & Bird Co., Chicago, IL). The animal is housed separately for 7 days for recovery before the subsequent operation. For icv cannulated animals, the correct position of the cannula is confirmed by a positive dipsogenic response to angiotensin II (Sigma, St Louis, MO). The correct cannula placement is also confirmed by histological examination after studies are completed in which the animal is infused with blue dye prior to sacrifice, and the brain is examined for ventricular staining. Only those animals with correctly positioned cannulae, independently assessed, are included in the study.

AgRP administration

[0053] Each animal is handled daily for 3 consecutive days prior to the initiation of the study, simulating the restraint used during the injection of the compound. Normal saline or 2 nmole of AgRP (84-132 amino acid fragment (Neurocrine Biosciences) is infused over 45 s in lateral ventricle-cannulated mice, using a 10-μl microsyringe (Hamilton Co., Reno, NE), every 3 days.

Leptin Administration

[0054] ob/ob mice are handled daily for 3 consecutive days prior to the initiation of the study, simulating the restraint used during the injection of the compound. Normal saline or 200 ng of recombinant leptin (Sigma Chemical) is infused over 45 s in lateral ventricle- cannulated mice, using a 10-μl microsyringe (Hamilton Co., Reno, NV), daily.

uCT Scanning

[0055] μCT scanning is used for non-destructive, three-dimensional evaluation of bone architecture. Right femurs are scanned using a Scanco™ μCT40 scanner (Scanco Medical AG) at a voxel size of 12x12x12 μm. Entire femurs (cancellous + cortical bone) are evaluated followed by site specific evaluation of cortical bone at the femoral midshaft and cancellous bone in the distal femur metaphysics. The threshold for analysis is determined empirically and set at 255 (0 - 1,000 range) for all evaluations. For the femoral midshaft, 20 slices (240 μm) of bone are evaluated, and total cross-sectional tissue volume (cortical and marrow volume, mm³) and cortical thickness (μm) are measured. For the femoral metaphysics, 120 (1.4 mm) slices were evaluated. Direct cancellous bone measurements included cancellous bone volume/tissue bone volume (%), trabecular number (1 mm), trabecular thickness (mm), and trabecular spacing (mm).

Blood chemistry analysis

[0056] After 6 weeks of observation, animals are sacrificed, and blood samples are collected for serum chemistry analysis. Blood urea nitrogen (BUN), serum creatinine and blood bicarbonate levels are assayed by standard laboratory methods. Serum leptin concentrations are measured with a mouse leptin radioimmunoassay kit (Linco Research, Inc., St Charles, MO). This method detects 0.2 ng/mL leptin. N-intact parathyroid hormone and 1,25 dihydroxyvitamin D3 concentrations are also measured, using commercially available kits.

Bone densitometry [0057] Bone mineral density (BMD) measurements are determined by dual energy X-ray absorptiometry (DEXA). Mouse whole body BMD measurements are obtained with a pencil beam Hologic QDR 1500 densitometer (Hologic, Waltham, MA). Densitometric analyses are performed on anesthetized mice. Food is withheld the night prior to examination (to eliminate confounding effects of undigested rodent chow on BMD assessment), and the mice are anesthetized with isoflurane inhalation. The animals are weighed to the nearest 0.1 g and immediately undergo DEXA scanning. The densitometer is calibrated daily with a hydroxyapatite phantom of the human lumbar spine. Analysis is performed using the mouse whole body software (version 3.2), from the manufacturer. The global window is defined as the whole body image minus the calvarium, mandible and teeth. The BMD values for the spine and right hip of the anesthetized animals is determined using the high-resolution software package on a QDR- 1500 bone densitometer (Hologic, Waltham, MA). BMD measurements of excised mouse femora is performed with a pixiMus densitometer (GE Medical Systems, Waukesha, WI). Routine calibration is performed daily with a defined standard.

Femoral shaft biomechanical testing

[0058] Mice are euthanized by CO₂ inhalation and weighed to the nearest 0.1 g. The left femoral bone is harvested immediately, wrapped in sterile gauze soaked in phosphate- buffered saline and stored frozen at -8O⁰C for subsequent analyses. Femora are tested for failure in three-point bending with a high-resolution materials test apparatus (Model 4442, Instron Corp., Canton, MA). The loading fixture consists of two fixed lower supports, placed at a span length (L) of approximately 7 mm, and an upper loading point attached to a moving actuator. The femur are placed with its posterior surface resting on the two lower supports. The upper loading point contacts the specimen at its mid-point, which is coincident with the center of the span. System software (Series IX for Windows 95, Instron Corp) is used to displace the actuator at the strain rate of 0.5%/sec until failure occurs. Load and displacement data is also collected using system software, and failure load and stiffness is determined.

Statistical analysis and sample size [0059] Data are expressed as means ±SE. Results are analyzed by Student's t-test when two groups are included or one-way ANOVA with post hoc analysis when three groups are included. Data sets are analyzed for statistical significance using SPSS™ 11.0 software package (SPSS Inc., Chicago, IL, USA). A sample size of 10 animals per group was used.

Example 2 -Bone Disease in Nephrectomized Wild-Type Mice

[0060] The present example demonstrates the utility of the present invention for providing treatment for bone loss in humans having a uremic metabolic disorder.

[0061] Eight- week old wild type (WT), male C57BL/6J mice were used in this study. Animals underwent 5/6 nephrectomy or sham operations and were fed 17% protein chow. Nephrectomized wild-type (WT-N) mice were uremic, but not acidotic. WT-N mice had higher BUN and creatinine (n=30, 48.6 ± 7.1 mg/dL; 0.54 ± 0.11 mg/dL) than WT-sham control (WT-S) (n=31, 26.4 ± 2.9 mg/dL; 0.23 ± 0.05 mg/dL) with pO.OOOl (Table 1). Blood bicarbonate levels were not different in WT-N mice (26.1 ± 0.6 mmol/L) versus WT-S mice (26.4 ± 0.4 mmol/L). All studies were performed over a 6-week period.

Table 1: Cumulative weight gain, cumulative food consumption, and serum chemistry of studied mice. Animals were sacrificed at the end of 6 weeks. Ten groups of animals are included: wild-type (WT) sham (S) versus nephrectomized (N); ob/ob-S versus ob/ob-N; MC4-RKO-S versus MC4-RK0-N; WT-N treated with AgRP (AgRP) versus WT-N vehicle control (V); ob/ob-N treated with Leptin (Lep) versus ob/ob-N/V . Data are expressed as means ± SE. ^#p<0.0001.

[0062] Before the initiation of the experiment and at the end of 6-week observation (prior to the sacrifice), in vivo whole body bone mineral density (BMD) measurements were carried out on anesthetized mice using the pixiMUS™ dual-energy X-ray absorptiometer (GE Medical System, Waukesha, WI) after an overnight fast. Analysis was performed using the mouse whole body software provided by the manufacturer. The BMD values of isolated femoral specimens were determined with the pixiMUS™ as well.

[0063] The geometry and microarchitecture of femoral specimens were examined with a desktop X-ray microtomographic scanner (Model UCT-40, Scanco, Wayne, PA). To determine femoral structural properties, each bone was tested for failure in three-point bending on a high-resolution materials test apparatus (Model 4442, Instron Corp., Canton,

MA). System software was used to displace the actuator at a strain rate of 0.5%/sec, until failure occurred. Load and displacement data were collected using system software to determine failure and stiffness.

[0064] WT-N mice were fed ad libitum (153.3 ± 5.6 g), whereas WT-S mice were pair- fed (153.3 ± 5.6 g; Table 1). WT-N mice gained less weight (gain of 0.3 ± 0.1 g) compared to pair-fed WT-S (gain of 2.1 ± 0.5 g; pO.0001; Table 1). As shown in Figure 1, WT-N mice exhibited a reduction of fat and lean mass whereas WT-S mice continued to gain both fat and lean mass. The fact that WT-N mice managed to gain total body weight despite reductions in fat and lean mass suggested fluid retention as a consequence of reduced renal function. [0065] The present results demonstrate the development of renal bone disease in uremic WT mice. There was no difference in initial whole body BMD between WT-N and WT-S mice (49.5 ± 3.4 mg/cm² versus 49.7 ± 1.5 mg/cm²; NS; Figure 1C). At the end of 6-week study, final whole body BMD tended to be lower in WT-N versus WT-S mice (48.8 ± 3.4 mg/cm versus 50.1 ± 1.6 mg/cm ; NS; Figure IB), although the difference did not reach statistical significance. However, further analysis indicated that WT-N mice experienced a decline in whole body BMD (loss of 1.24±0.44%) while pair- fed WT-S mice gained whole body BMD (gain of 0.70 ± 0.33%; pO.OOOl; Figure IB). Moreover, there was evidence of impaired linear growth due to the uremic state. Femoral lengths after 6 weeks of uremia were significantly lower than sham-operated pair-fed controls (15.4 ± 0.06 versus 15.2 ± 0.05 mm; p<0.04).

[0066] Consistent with the decline in whole body BMD, WT-N mice also exhibited a significant reduction in femoral BMD (42.2 ± 0.3 versus 48.6 ± 0.3 mg/cm ; p<0.0001; Figure 2B). The reduced femoral BMD appeared to be a consequence of impaired bone development. Uremic WT mice had 11% reduced cortical bone area (Figure 2C) and 15% reduced cortical thickness (Figure 2D) in comparison to pair-fed WT-S mice. As a consequence of the reduced bone density and bone geometry, the biomechanical strength (as measured by failure load) was reduced by 21% (11.1 ± 2.5 versus 14.2 ± 1.5 N; pO.OOOl; Figure 2E) when compared with pair-fed WT-S mice. Thus, this murine model of uremia faithfully recapitulates many of the skeletal features of renal disease observed in growing children. A set of examples are demonstrated in Figure 4.

Example 3 - ob/ob Mice Resist Uremic Bone Disease

[0067] The present example demonstrates that the resistance of ob/ob mice to uremic bone disease.

[0068] Leptin is a powerful inhibitor of bone formation in vivo. The role of leptin in the development of renal bone disease was tested using ob/ob (leptin deficient) mice. These mice were ordered from Jackson Laboratories and had the same C57BL/6 genetic background as the WT mice used in the present studies. [0069] Both ob/ob-N (n=23) and ob/ob-S (n=21) were pair-fed with WT-N mice (Table 1). All male animals were used. Pair- fed ob/ob-N mice had higher BUN and creatinine levels (64.1 ± 5.5 mg/dL and 0.60 ± 0.10 mg/dL, respectively) compared with those in ob/ob-S mice (29.0 ± 3.6 mg/dL; 0.20 ± 0.10 mg/dL, respectively, p<0.0001), and were not acidotic (Table 1). Blood bicarbonate levels in ob/ob-N (26.3 ± 0.3 mmol/L) and ob/ob-S (26.4 ± 0.3 mmol/L; NS; Table 1) were normal. [0070] There was no difference in weight gain between ob/ob-N and ob/ob-S mice (gain of 2.3 ± 0.2 g versus 2.3 ± 0.3 g; NS; Table 1) and body composition (lean and fat mass; Figure 1, D and E) was unaffected by uremia in ob/ob mice. Moreover, there was no significant difference in any skeletal parameter (whole body BMD, femoral length, femoral BMD, femoral geometry or femoral failure load) between ob/ob-N mice and pair-fed ob/ob-S mice (Figure ID and Figure 2). Thus, renal bone disease did not develop in nephrectomized ob/ob mice.

Example 4 - MC4-RKO mice Resist Uremic Bone Disease

[0071] Leptin exerts its hypothalamic effects through the melanocortin-4 receptor (MC4- R). The role of central leptin studies in the development of uremic bone disease was therefore tested using MC4-R knockout (KO) mice. These mice had the same C57BL/6 genetic background as the WT mice used in our experiments. MC4-R KO mice underwent 5/6 nephrectomy or sham operation and were fed 17% protein chow. Both MC4-R KO-N (n=30) and MC4-R KO-S (n=31) were pair-fed with WT-N mice (Table 1). All male animals were used. MC4-R KO-N mice had higher BUN and creatinine levels (61.8 ± 6.6 mg/dL and 0.56 ± 0.08 mg/dL, respectively) than MC4-R KO-S mice (25.0 ± 2.4 mg/dL; 0.23 ± 0.05 mg/dL, respectively, p<0.0001) and were not acidotic (Table 1). Blood bicarbonate levels in MC4- RKO-N (26.4 ± 0.3 mmol/L) and in MC4-R KO-S mice (26.5 ± 0.4 mmol/L; NS; Table 1) were normal. [0072] There was no difference in weight gain between MC4-R KO-N and MC4-R KO-S mice (gain of 3.1 ± 0.6 g versus 4.0 ± 0.5 g; NS; Table 1). As expected, MC4-R KO mice exhibited increased fat mass (compared to WT), but body composition (lean and fat mass; Figure 1, D and E) was unaffected by uremia. As observed in the ob/ob mouse model, there was no significant difference in any skeletal parameter (whole body BMD, femoral length, femoral BMD, femoral geometry or femoral failure load; Figure ID and Figure 2) between MC4-R KO-N mice and pair-fed MC4-R KO-S mice. Thus, renal bone disease did not develop in nephrectomized MC4-R KO mice. [0073] The mouse model of uremic bone disease used herein is characterized by reduced bone mass and bone strength - features identical to those observed in humans (especially adolescents) with chronic renal disease. The preservation of bone mass, bone strength in the face of uremia in two mouse models with deficient leptin action strongly implicates aberrant leptin signaling as an important determinant of uremic bone disease. The present findings in mice provide proof of principle in pinpointing pathways (central and/or peripheral) for focused human therapeutic drug development and the development of new therapeutic approaches for uremic bone disease. These findings also offer important insight into the skeletal physiological mechanisms impacted by kidney disease.

Example 5 - AgRP Administration reversed Cachexia and Prevented Hvperleptinemia in Uremic WT Animals

[0074] The present example is provided to demonstrate the utility of the present invention for reducing bone degradation symptoms observed in uremic animals (such as reduced bone density and weight), and to show that these bone-related effects are directly related to the level of leptin in an afflicted animal. The present example also demonstrates that manipulation of leptin levels in a uremic animal can be used to control and/or halt bone degradation symptoms in uremic animals. [0075] The effect of melanocortin receptor blockade in our animal model of uremic cachexia was investigated using a pharmacological approach. AgRP, produced in the arcuate nucleus, is an antagonist of MC4-R. The response to central administration of AgRP in WT- N mice was examined. Nephrectomized WT mice were treated with 2 nmol of AgRP (84- 132 amino acid fragment, Neurocrine Biosciences, San Diego, CA), infused over 45 seconds in lateral ventricle-cannulated mice, using a 10-μl syringe. During 14 days of preliminary study, experimental animals were infused with either normal saline or AgRP at days 0, 3, 6, 9, and 12 relative to the initial injection.

[0076] AgRP-treated WT-N (WT-N/AgRP, n=14) and WT-N (n=14) mice were uremic but not acidotic. WT-N/AgRP and WT-N mice had higher BUN (58.4±9.2 mg/dL; 54.1 ± 6.3 mg/dL) and creatinine (0.49 ± 0.1 mg/dL; 0.49 ± 0.1 mg/dL) compared to WT-S mice (n=14, 29.1 ± 8.1 mg/dL; 0.26 ± 0.1 mg/dL) (ANOVA, pO.0001). Blood bicarbonate levels were not different in WT-N/AgRP (24.9 ± 1.7 mg/dL) and WT-N (25.4 ± 2.4 mg/dL) versus WT- S mice (25.7 ± 2.1 mg/dL). [0077] Cachexia was reversed by AgRP treatment in the herein cited JCI paper (11) by the present inventors. Furthermore, while adjusted serum leptin levels were still high in WT- N mice (3.56 ± 0.96 ng/mL) (ANOVA, p<0.0001), administration of AgRP significantly decreased the serum leptin level in the uremic animals. There was no difference in the adjusted serum leptin levels between WT-N/AgRP and WT-S mice (2.42 ± 0.76 ng/mL; 2.21 ± 0.40 ng/mL) (Figure 3).

Example 6 - Leptin Signaling in the Pathogenesis of Uremic Bone Disease

[0078] The present example demonstrates the effect of central (intracerebroventricular) leptin replacement on bone mass in uremic ob/ob mice.

[0079] Both ob/ob-N/Lsp (n=7) and ob/ob-N/Y (n=8) were pair-fed with WT-N mice (Table 1). All male mice were used. Both ob/ob-N/Lep and ob/ob-N/Y mice had higher BUN and creatinine levels (62.1±2.9 mg/dL and 66.0±3.7; 0.60±0.10 mg/dL and 0.60±0.30; respectively) compared with those in ob/ob-S mice (p<0.0001) and were not acidotic. [0080] Lev. leptin administration induced significant bone loss in ob/ob-N/Lep mice while ob/ob-N/Y mice resisted uremic bone loss. ob/ob-N/Lep lost weight (-7.3±0.6 g) while ob/ob-N/Y continued to gain weight (2.8±0.2 g; p<0.0001, Table 1). ob/ob-N/Lep exhibited a reduction of fat mass (loss of 51.4±3.3%) whereas ob/ob-N/Y gained fat mass (gain of 4.7+1.7%; p<0.0002, Figure IE). Both ob/ob-N/Lep and ob/ob-N/Y mice gained lean mass (gain of 4.6±0.5% versus gain of 3.8±0.6%; NS, Figure ID). Moreover, further analysis indicated that ob/ob-N/Lep mice lost whole body BMC (loss of 2.5±0.6%) while ob/ob-N/Y mice continued to gain whole body BMC (gain of 2.4±0.4%, p< 0.0001, Figure IB).

Example 7 - Central leptin signaling blockade by a melanocortin receptor antagonist (AgRP) and Effect on Bone Mass and Biomechanical Strength in Uremic WT Mice

[0081] The present example demonstrates the impact of central and peripheral pharmacologic blockade of leptin signaling on uremic bone disease. More specifically, the present example demonstrates the utility of the present invention for regulating bone mass through leptin signaling at the MC4-R. [0082] MC4-R KO mice display an increase in bone mass (26). MC4-R deficient patients have an increased bone mineral density (4). The results generated by the present inventors showed that renal bone disease did not develop in nephrectomized MC4-R KO mice. There was no difference in initial whole body BMD or final whole body BMD between MC4-R KO-N and MC4-R KO-S mice. In addition, there was no difference in whole body BMD gain between MC4-R KO-N and MC4-R KO-S mice. There was no difference in femoral BMD, cortical area or failure load between the MC4-R KO-N mice than pair-fed MC4-R KO-S mice. There was also no difference in weight gain between MC4-R KO-N and MC4-R KO-S mice. From this, the present inventors have demonstrated that blocking the central leptin signaling effect on bone mass through MC4-R prevents the development of renal bone disease.

[0083] Data has been generated by the present inventors showing that MC4-R KO mice do not exhibit the phenotype of cachexia despite induction of uremia (11). In order to control for the impact of nutrition intake, the nephrectomized MC4-RK0 mice will be pair- fed to the uremic wild-type (WT) mice. So the experimental groups will include, uremic WT mice fed ad libitum, sham WT, uremic MC4-R KO and sham-operated MC4-R KO. The latter three groups will be pair-fed with uremic WT group. Intracerebroventricular (icv) administration of AgRP, a natural antagonist of both MC4-R and MC3-R, will be examined to determine if it can reverse the phenotype of renal bone disease in WT uremic mice. As presented herein, corrected leptin levels are normalized after 14 days of AgRP treatment. The mechanism of this normalization will also be examined. AgRP treatment will be monitored to demonstrate that this treatment prevents renal bone disease with normalization of serum leptin concentrations as part of the outlined 6 week study.

[0084] β2-adrenergic receptor deficient mice have the HBM phenotype. Isoproterenol, a β2-adrenergic agonist, has been shown to prevent the HBM in ob/ob mice, which are leptin deficient. Propranolol, an adrenergic blocker, prevents and blocks the effect of isoproterenol on osteoblasts (4). Propranolol also increases bone mass in WT and ovariectomized mice (35). Propranolol will be examined to determine if it will block the effect of increased leptin signaling on bone in nephrectomized WT mice. [0085] Intracerebroventricular (icv) AgRP is demonstrated here to reverse anorexia and cachexia in WT mice. To control for nutritional effects, the AgRP treated uremic WT mice will also be pair-fed with uremic WT mice. In order to realize the full potential of the beneficial effects of AgRP in reversing both cachexia and renal bone disease, two further groups will be created and examined, one of these groups being AgRP treated uremic mice fed ad libitum, and another group being uremic mice forced fed to match the intake of the former group.

[0086] Intracerebroventricular (icv) infusions may result in leakage of AgRP into the systemic circulation. There are MC4-R receptors in various organs including bone (36,37). There is evidence of direct actions of melanocortin peptides on bone metabolism (37). AgRP, an antagonist of MC4-R, may have direct peripheral effects. AgRP concentrations will therefore be monitored during these studies to ensure there is no leakage.

[0087] AgRP is a hypothalamic neurotransmitter that antagonizes both MC3-R and MC4- R. AgRP has only been shown to be effective when it is given icv and this route of administration limits its therapeutic potential in humans. However, specific MC4-R anatagonists are now available which may be given peripherally (38). These compounds will be tested in the present mouse model of renal bone disease.

[0088] The present example demonstrates the specificity of the present treatment methods for providing a method that is essentially free of regulatory effects at the MC3 receptor.

[0089] MC3-R KO mice will be examined in the present example to examine the role of MC3-R in leptin signaling in the context of renal bone disease. Experiments as described in example 4 above will be performed in the MC3-R KO mice.

Example 8 - Screening Method for Selection of MC4 Antagonists

[0090] The present example demonstrates the utility of the present invention as providing for a screening method useful in the selection of materials that may be used in the preservation of bone health in a uremic animal. By way of example, the agouti-related peptide (AgRP) may be used as the baseline compound against which other candidate compounds from a selected library of commercially available compounds will be compared and selected against.

[0091] An MC4-R antagonist drug discovery program will be initiated by utilizing information derived from structure-activity relationships of MC4-R agonists and mutagenesis results of the MC4-R and peptide ligands (such as alpha melanocyte-stimulating hormone (MSH), NDP-MSH and MTII). [0092] In vitro binding and antagonist and agonist activities at the human MC4-R will be tested. For instance, the synthesized compounds will be tested for their ability to compete with radiolabeled NDP-MSH in cells. By way of example, a cell line that expresses the MC4 receptor that may be used in the present invention is the HEK 293 cell line. The HEK 293 cell line is available from commercial sources.

[0093] As part of the selection and screening protocol, the IC50 and Ki of the candidate MC4 receptor antagonist or library of candidate antagonists, will be determined. Compounds that possess a Ki value of about 1.8 nM or more in binding affinity for the MC4 receptor may be selected as part of at least an initial step in the screening process. Selected compounds will be measured to determine their ability to stimulate cAMP in the same cell lines similarly engineered to express the MC4 receptor.

[0094] The specificity of each candidate compound tested for binding to MC4-R may also be tested against other MC receptors 1, 2, 3 and 5 to detect potential interactions and side-effects, as well as specificity for each one of the MC receptors. Preferably, candidate compounds that are selected will exhibit enhanced specificity for the MC-4 receptor, and less for other of the MC receptor types (e.g., 1, 2, 3 or 5).

[0095] The screening method may comprise exposing a control culture of cells expressing an MC4 receptor to a known agonist, such as melanocyte stimulating hormone (MSH) or melanotan (MT) II, to provide an activated culture of cells. The activated culture of cells is then exposed to a known antagonist or reverse agonist of MC4 receptor, such as agouti related protein (AgRP), to provide a control measure of MC4 receptor inhibitory activity. For purposes of this screening method, the control inhibitory compound, such as AgRP, is considered to produce a hypothetical 100% inhibition of the MC4 receptor. Separately, an activated test culture of cells expressing an MC4 receptor and previously exposed to the known agonist is exposed to a set of candidate substances with potential antagonist or reverse agonist activity. Candidate substances are selected if they have a measure of melanocortin-4 receptor inhibitory activity of about 50% or more compared to the control inhibitory compound. Compounds selected as a result of this initial in vitro or in cellulo screen may then be tested for their clinical potential in preventing or treating preexisting bone disease using the uremic mouse model described herein.

[0096] In another aspect of the screening method, compounds having specific activity for the MC3 receptor will be obtained. In some of these embodiments, the method comprises exposing a control culture of cells comprising cells expressing a melanocortin 3 receptor to a MC3-R agonist to provide an activated control measure of MC3-R activity; exposing a second test culture of activated cells expressing the MC3-R to a candidate substance and obtaining a measure of the candidate substance agonist activity and selecting a candidate substance demonstrating a MC3-R agonist activity that provides at least 50% or more of the control substance MC3-R agonist activity. In some embodiments, this method may be described as providing selected compounds that are essentially free of melanocortin 4 receptor specific activity. In some embodiments, the MC3-R agonist is D-Trp⁸-γ-MSH. A further step that may be included in some embodiments of this method would include an in vivo screening of the selected candidate compound using the mouse model for uremia described herein.

[0097] Alternatively, candidate compounds that are selected will exhibit enhanced specificity for the MC-3 receptor, and less for other of the MC receptor types (e.g., 1, 2, 4 or 5). As a second step to the screening protocol, a candidate compound screened and determined to have acceptable activity for inhibition of melanocortin receptor on the above described cell line, will be further tested in an in vivo model, such as the animal model for renal osteodystrophy developed by the present inventors.

Example 9 - Examples of MC4-R Antagonists [0098] The present example presents additional MCR receptor antagonists that will be used in the practice of the present invention. Some specific MC4 receptor antagonists are described in Markinson et al. (2005) (38), which reference is specifically incorporated herein by reference in its entirety. By way of example, this reference includes: 4-{(2R)-[3- Aminopropionylamido]-3-(2,4-dichlorophenyl)propionyl}-l-{2-[2-thienyl) ethylaminomethyl] phenyl} piperazine (See Chen C et al, J Med Chem 47:6821-30, 2004); AgRP, which has been described in our initial data is actually a reverse agonist and inhibits both MC4-R as well as MC3-R. (See Adan RAH et al, (2006), Br J Pharmacology 149:815-827); SHU9119; HSO 14 ; Corticotropin-releasing hormone (CRH) antagonist: alpha helical-CRH(9-41 ) .

[0099] Other compounds that may be used in the practice of the present invention may be selected based on relative activity for activity as an MC4 receptor antagonist compared to, for example, Agouti related peptide (AgRP). In addition, the compounds named above may be used instead of the agouti-related peptide as a standard compound to be used in the screening method as described above.

[00100] All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference. Although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom. Bibliography

The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach a methodology, techniques, and/or compositions employed herein.

I) Ducy P, et al (2000), Cell, 100: 197-207. 2) Elefteriou F, et al (2004), Natl Acad Sci USA, 101:3258-63.

3) Cumin F, (1997), Int Jobes Relat Metab Disord, 21 :495-504, 1997.

4) Daschner M, et al (1988), J Am Soc Nephrol, 9: 1074-9.

5) Sharma K, et al (1997), Kidney Int, 51 : 1980-5.

6) Coen G, et al (2003), Am J Kidney Dis, 42: 1036-42, 7) Mak RH, et al (1985), British Medical Journal, 291:623-7.

8) Turner, et al (1988), Kidney Int, 33:989-95.

9) Mak RH. (1992), Kidney Int, 41: 1049-54.

10) Mak RH. (1998), Kidney Int, 53: 1353-7.

I I) Cheung W, et al (2005), J. Clin Invest, 115: 1659-65. 12) Mitch WE. (2005), J Clin Invest, 115: 1476-8.

13) Ritz E. (2005) J Am Soc Nephrol, 16:2245-50.

14) Wiecek A. (2005) Nephrol Dial Transpl, 20:2620-2.

15) Klein RF, et al (2004), Science, 303:229-32.

16) Consensus Development Conference: Prophylaxis and treatment of osteoporosis. Am J Med, 646-50, 1993.

17) Riggs B, Melton LJI. (1986), N Engl J Med, 314: 1676-8.

18) Riggs B, et al (1998), J Bone Miner Res, 13:763-73. 19) Felson DT, et al (1993), J Bone Miner Res, 8:567-73.

20) Tremollieres FA, Pouilles JM, Ribot C. (1993), J Clin Endocrinol Metab, 77:683-6.

21) Ravn P, Cizza G, Bjarnason NH et al. (1999), J Bone Miner Res, 14: 1622-7.

22) Brown SA, Sharpless JL., (2004), Clin Diabetes, 22: 10-20. 23) Taal MW, Masud T, Green D, Cassidy MJD. (1999), Nephrol Dial Tranpl, 14: 1922- 28.

24) Atsumi K, et al (1999), Am J Kidney Dis, 33:287-93, 1999.

25) Hutchinson AJ, et al (1993), Kidney Int, 44: 1071-7.

26) Orwoll B, Bouxesein ML, Marks DL, Cone RD, Klein RF. In ORS/AAOS Presentations 2003, 71^st Annual Meeting of AAOS (ORS, San Francisco, CA 2004).

27) Cone RD. (2005), Nature Neurosci, 8:571-8, 2005.

28) Matsunuma A, et al. (2004), Endocrinology, 145: 1367-75.

29) Farooqi IS, et al (2000), J Clin Invest 106:271-9.

30) Khosla S, et al (1996), J Bone Miner Res, 11 :857-63. 31) Winters K, Snow C. (2000), J Womens Health Gend Based Med, 9:865-72.

32) Young D, et al (2001), Osteoporosis Int, 12:506-15.

33) Steppan Cm, et al (2000), Regulatory peptides, 92:73-78.

34) Elefteriou F, et al (2005), Nature, 434:514-20.

35) Takeda S, et al (2002), Cell, 111:305-17. 36) Mountjoy KG, et al (2003), Endocrinology, 144:5488-96.

37) Dumont LM, et al (2005), Peptides, 26: 1929-

38) Cheung W, et al (2005), J Clin Invest, 115: 1659-65.

39) Chen, et al (2004), J. Med. Chem., 47: 6821-6890.

40) Marks DL et al (2006) Peptides 27(2):259-264.

Claims

What is claimed is:

1. A screening method to identify substances suitable for the treatment of bone loss or osteodystrophy accompanying renal disease in a mammal comprising:

exposing a culture of cells expressing a melanocortin-4 receptor to a known melanocortin-4 receptor agonist to provide an activated culture of cells for melanocortin- 4 receptor activity;

separating the activated culture of cells into at least two activated cultures of cells, including a first activated culture of cells and a second activated culture of cells;

exposing the first activated culture of cells to a known melanocortin-4 receptor antagonist or reverse agonist to provide a control measure of 100% inhibition of melanocortin-4 receptor activity;

exposing the second activated culture of cells to a candidate substance; and

selecting a candidate substance that provides at least 50% or more inhibition of melanocortin-4 receptor activity compared to the known antagonist or reverse agonist.

2. The screening method of claim 1, wherein the substance is further selected based on its ability to reduce bone loss or osteodystrophy in an in vivo mouse model.

3. The screening method of claim 1, wherein the known melanocortin-4 receptor agonist is melanocyte stimulating hormone (MSH) or melanotan (MT) II.

4. The screening method of claim 1 , wherein the known melanocortin-4 receptor antagonist or reverse agonist is Agouti-related protein or peptide (AgRP).

5. A screening method to identify substances suitable for the treatment of bone loss or osteodystrophy accompanying renal disease in a mammal comprising:

exposing a first culture of cells expressing a melanocortin-4 receptor to a known melanocortin-4 receptor agonist to provide a first activated culture of cells for melanocortin-4 receptor activity; exposing the first activated culture of cells to a known melanocortin-4 receptor antagonist or reverse agonist to provide a control measure of 100% inhibition of melanocortin-4 receptor activity;

exposing a second culture of cells expressing a melanocortin-4 receptor to a known melanocortin-4 receptor agonist to provide a second activated culture of cells for melanocortin-4 receptor activity;

exposing the second activated culture of cells to a candidate substance; and

selecting a candidate substance that provides at least 50% or more inhibition of melanocortin-4 receptor activity compared to the known antagonist or reverse agonist.

6. The screening method of claim 6, wherein the substance is further selected based on its ability to reduce bone loss or osteodystrophy in an in vivo mouse model.

7. The method of claim 6, wherein the known melanocortin-4 receptor agonist is melanocyte stimulating hormone (MSH) or melanotan (MT) II.

8. The method of claim 6, wherein the known melanocortin-4 receptor antagonist or reverse agonist is Agouti -related protein or peptide (AgRP).

9. A screening method to identify substances suitable for the treatment of bone loss or osteodystrophy accompanying renal disease in a mammal comprising:

exposing a culture of cells expressing a melanocortin-4 receptor to a detectably labeled ligand for melanocortin-4 receptor and a candidate substance; and

selecting a candidate substance that is capable of competing with the detectably labeled ligand for binding to the melanocortin-4 receptor, thus reducing the amount of detectably labeled ligand bound to the receptor.

10. The screening method of claim 9, wherein the ligand is radiolabeled NDP-MSH.

11. A screening method to identify substances suitable for the treatment of bone loss or osteodystrophy accompanying renal disease in a mammal comprising: exposing a culture of cells expressing a melanocortin-3 receptor to a known melanocortin-3 receptor agonist to provide an activated control measure of melanocortin- 3 receptor activity representing 100% activation;

exposing a second culture of cells expressing a melanocortin-3 receptor to a candidate substance; and

selecting a candidate substance demonstrating a melanocortin-3 receptor activation at least 50% compared to the control measure of melanocortin-3 receptor activation.

12. The screening method of claim 13, wherein the substance is further selected based on its ability to reduce bone loss or osteodystrophy in an in vivo mouse model.

13. The method of claim 13, wherein the known melanocortin-3 receptor agonist is D- Trp⁸-γ-MSH.

14. A pharmaceutical composition for the treatment of bone loss or osteodystrophy accompanying renal disease in a mammal comprising a therapeutically effective amount of a substance identified by the screening methods of claims 1 , 5, 9, or 11 , further comprising a pharmaceutically acceptable carrier.

15. A method for reducing bone loss or osteodystrophy accompanying renal disease in a mammal comprising administering a therapeutically effective amount of a substance identified by the screening methods of claims 1, 5, 9, or 11.