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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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  • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates generally to bone physiology. More specifically, the present invention relates to in vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction.
• Bone loss due to glucocorticoid excess is diffuse, affecting both cortical and cancellous bone, but has a predilection for the axial skeleton.
• Spontaneous fractures of the vertebrae or ribs are, therefore, often presenting manifestations of the disorder (3,4).
• a cardinal feature of glucocorticoid-induced osteoporosis is decreased bone formation (5).
• patients receiving long-term glucocorticoid therapy sometimes develop collapse of the femoral head (osteonecrosis), but the mechanism underlying this is uncertain (6).
• Defective osteoblastogenesis has been reported to be linked to reduced bone formation and age-related osteopenia in the S AMP6 mouse (7). Besides the relationship between aberrant osteoblast production and osteoporosis, it has been recently shown that a significant proportion of osteoblasts undergo apoptosis (8), which raises the possibility that the premature or more frequent occurrence of osteoblast apoptosis could contribute to incomplete repair of resorption cavities and loss of bone.
• the prior art is deficient in compounds that possess the advantageous properties of glucocorticoids, namely anti-inflammatory properties, but do not cause bone loss o r osteoporosis.
• the present invention provides for methods of screening compounds to fulfill this long-standing need in the art.
• glucocorticoid-induced bone disease is due to changes in the birth or death rate of bone cells
• a murine model of glucocorticoid excess was used as well as bone biopsy specimens obtained from patients with glucocorticoid- induced osteoporosis.
• This invention demonstrates that glucocorticoid administration decreases bone formation rate an d bone mineral density accompanied by defective osteoblastogenesis and osteoclastogenesis in the bone marrow and increases apoptosis of mature osteoblasts and osteocytes.
• One object of the present invention is to provide methods to screen compounds that retain the anti-inflammatory properties of glucocorticoids yet do not result in bone loss o r osteoporosis due to apoptosis of osteoblasts and osteocytes.
• a method of screening for compounds that reduce th e bone deteriorating effects of glucocorticoids comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid alone or a glucocorticoid in combination with a test compound; and (b) comparing the number of cells undergoing apoptosis following treatment with the glucocorticoid alone o r following treatment with the glucocorticoid in combination with the test compound; wherein a lower number of apoptotic cells following treatment with the glucocorticoid in combination with the test compound than with the glucocorticoid alone indicates that the test compound reduces the
• This embodiment also includes the aforementioned method, wherein the compound has little effect on the anti-inflammatory properties of the glucocorticoid, further comprising the step of comparing the anti-inflammatory response of the glucocorticoid in combination with the test compound to the anti-inflammatory response of the glucocorticoid alone; wherein essentially equivalent anti-inflammatory responses of th e glucocorticoid alone and the glucocorticoid in combination with th e test compound is indicates that the test compound both reduces the bone deteriorating effects, while retaining the anti- inflammatory properties of the glucocorticoid; wherein said anti- inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.
• a method of screening for glucocorticoid analogs th at possess decreased apoptotic properties towards osteoblast an d osteocyte cells comprising the steps of: (a) contacting the cells with either a glucocorticoid or a glucocorticoid analog; and (b) comparing the number of apoptotic cells following treatment with the glucocorticoid or the glucocorticoid analog, wherein a lower number of apoptotic cells following treatment with the glucocorticoid analog than with the glucocorticoid indicates that the glucocorticoid analog possesses decreased apoptotic properties towards the cells.
• This embodiment also includes th e aforementioned method, wherein the glucocorticoid analog retains anti-inflammatory properties, further comprising the step of: (c) comparing the anti-inflammatory response of the glucocorticoid in combination with a test compound to the anti-inflammatory response of the glucocorticoid alone, wherein essentially equivalent anti-inflammatory responses of the glucocorticoid alone and the glucocorticoid in combination with the tes t compound is indicative of a glucocorticoid analog that possesses decreased apoptotic properties while retaining anti-inflammatory properties; wherein said anti-inflammatory response is determined by models of inflammation selected from the group consisting of the adjuvant-induced arthritis model and hindlimb inflammation model.
• a method of screening for compounds that stimulate bone development comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and (b) comparing the nu mb er of cells undergoing apoptosis following treatment with th e glucocorticoid or the test compound; wherein a lower number of apoptotic cells following treatment with the test compound th an with the glucocorticoid indicates that the test compound stimulates bone development.
• a method of screening for compounds that increase bone mineral density comprising the steps of: (a) contacting osteoblast and osteocyte cells with either a glucocorticoid or a test compound; and (b) comparing the numb er of cells undergoing apoptosis following treatment with th e glucocorticoid and the test compound; wherein a lower number of apoptotic cells following treatment with the test compound than with the glucocorticoid is indicative of a compound that increases bone mineral density.
• contacting is selected from the group consisting of in vitro cell cultures and in vivo murine animal model and determination of apoptosis is selected from the group consisting of TUNEL, DNA fragmentation and immunohistochemical analysis.
• Figure 1 shows photomicrographs of the effects of prednisolone on murine vertebral cancellous bone.
• panel A is a longitudinal, panoramic section from a mouse receiving placebo and in panel B , a section from a mouse receiving prednisone.
• the histomorphometric reading area is outlined.
• Figure 2 shows quantification of CFU-OB an d osteoclast progenitors formed in ex vivo bone marrow cell cultures.
• Marrow cells were obtained from the femurs of male mice after 27 d of exposure to placebo (white bars) or 2. 1 mg/kg/d of prednisolone (black bars). Cells from each mou se were cultured separately.
• Figure 3 shows the effect of prednisolone on murine osteoblast apoptosis. Osteoblasts were counted in undecalcified sections of cancellous bone from the vertebral secondary spongiosa. In panel A , the placebo group is shown and in panel B, the higher dose prednisolone group. Apoptotic cells in this experiment were identified using TUNEL and morphometric features such as nuclear fragmentation and condensation of chromatin (arrows). Methyl green counterstain viewed with Nomarski differential interference microscopy, original magnification X400.
• Figure 4 shows the effect of prednisolone on murine osteocyte apoptosis.
• the cells were counted in undecalcified sections of femoral metaphyseal cortical bone.
• panel A th e placebo group is shown and in panel B, the higher dose prednisolone group.
• Apoptotic osteocytes (arrowheads) are seen in close proximity to normal cells.
• Methyl green counterstain viewed with Nomarski differential interference microscopy, original magnification X630.
• Figure 5 shows the effect of chronic prednisone treatment on apoptosis in human bone.
• TUNEL-positive osteoblasts (arrowheads) and osteocytes (arrows) were absent from normal subjects ( Figure 5 A ) but were clearly identified i n patients with prednisone-induced osteoporosis ( Figure 5B and Figure 5C).
• Approximately 5% of the osteocytes and 30% of th e osteoblasts were apoptotic.
• the photomicrographs are from transiliac bone biopsy specimens. Methyl green counterstain viewed with Nomarski differential interference microscopy, original magnification X630.
• Glucocorticoid-induced bone disease is characterized by decreased bone formation and in situ death of isolated - segments of bone (osteonecrosis) suggesting that glucocorticoid excess, the third most common cause of osteoporosis, may affect the birth or death rate of bone cells thus reducing their numbers .
• prednisolone was administered to 7 -month-old mice for 27 days and decreased bone density, serum osteocalcin and cancellous bone area along with trabecular narrowing w ere found.
• mice exhibited a 3-fold increase in osteoblast apoptosis in vertebrae and showed apoptosis in 28% of the osteocytes in metaphyseal cortical bone. As in mice, an increase in osteoblast and osteocyte apoptosis was documented in patients with glucocorticoid-induced osteoporosis .
• the present invention is directed towards methods of screening compounds that retain the anti-inflammatory properties of glucocorticoids while lacking the bone degeneration properties associated with long-term administration due to apoptosis of osteoblasts and osteocytes.
• the present invention is further directed towards methods of screening compounds that promote bone regeneration by inhibiting the apoptosis of osteoblasts and osteocytes.
• glucocorticoid and “glucocorticoid analog” is defined as substances that bind to th e glucocorticoid receptor.
• apoptosis refers to programmed cell death with nuclear fragmentation and cell shrinkage as detected by morphological criteria and Terminal Uridine Deoxynucleotidal Transferase Nick End Labeling (TUNEL) staining.
• anti-inflammatory response refers to preventing the induction of cytokines and other events that lead to T cell activation.
• cytokines cytokines and other events that lead to T cell activation.
• Several models of inflammation are routinely used in the art, including the adjuvant-induced arthritis model an d hindlimb inflammation model which are well known to those having ordinary skill in this art (54, 55).
• bone mineral density refers to bone mass as defined by Dual-Energy X-Ray Absorbtiometry (DEXA).
• DEXA Dual-Energy X-Ray Absorbtiometry
• mice Male Swiss Webster mice (Charles River Laboratories, Stone Ridge, NY) were electronically tagged (Biomedic Data System Inc., Maywood, NJ) and kept in plastic cages (3-5 animals p er cage) under standard laboratory conditions with a 12 hr dark, 1 2 hr light cycle and a constant temperature of 20°C and humidity of 48%. All mice were fed on a standard rodent diet (Agway RMH 3000, Arlington Heights, IL) containing 22% protein, 5% fat, 5% fiber, 6% ash, 3.5 Kcal/g, 1.0 IU vitamin D3/g, 0.97% calcium an d 0.85% phosphorus with water ad libitum. The animals and food supply were weighed at one week intervals throughout the experiment. Studies were approved by the UAMS Division of Laboratory and Animal Medicine.
• Dual-energy X-ray absorptiometry was used to determine global (whole body minus the head), spinal an d hindquarters bone mineral density in live mice (7,9).
• the scans done at 27 days after pellet implantation were analyzed using th e 'Compare' technique, in which the evaluation is based on the exact positioning and region of interest placement of the baseline scan.
• Serum osteocalcin was measured b y radioimmunoassay using a goat anti-murine osteocalcin an d murine osteocalcin as tracer and standard (Biomedical Technologies, Stoughton, MA).
• Urinary free deoxypyridinoline excretion was determined by a microtiter competitive enzyme immunoassay (Pyrilinks-D, Metra Biosystems, Mountain View, CA) and was expressed as a ratio to the urinary creatinine.
• the distal femora and lumbar vertebrae were fixed in 4°C Millonig's phosphate-buffered 10% formalin, pH 7.4, embedded undecalcified in methyl methacrylate and stained (7,9,13).
• the histomorphometric examination was done with a computer and digitizer tablet (OsteoMetrics Inc. Version 3.00, Atlanta, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Inc., Thornwood, NY) with a drawing tube attachment. All cancellous measurements were two-dimensional, confined to the secondary spongiosa and made at X400 magnification (numerical aperture 0.75).
• the terminology and units used are those recommended b y the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (14). The trabecular width and osteoid width were measured directly. Trabecular spacing and number were calculated (15). Only TRAPase-positive cells were included in the osteoclast perimeter. The rate of bone formation ( ⁇ m 2 / ⁇ m/d) and turnover (%/d) were calculated (7).
• CFU-F colony-forming unit-fibroblast
• CFU-OB CFU-osteoblast
• Osteoclast formation in bone marrow cultures w as assessed in replicate cultures (4-6 from each animal) maintained for 9 days in the presence of ⁇ MEM, 10% FBS and 10 nM 1.25(OH) 2 D 3 (7).
• marrow cells were cultured at 1.5 x 10 6 per 2 cm 2 well on 13 mm round Thermanox disks and maintained for 8 days in the presence of 10% FBS in ⁇ MEM supplemented with 10 "8 M 1.25(OH) 2 D 3 (provided by Dr. Milan Uskokovic, Hoffman-LaRoche, Nutley, NJ).
• the number of CFU-F colonies, CFU-OB colonies, an d osteoclastic cells formed from the marrow cells of each animal w a s expressed as the number per femur, which was calculated b y multiplying the number of colonies or osteoclasts obtained per 1 0 6 cells seeded at the initiation of the cultures by the total number of marrow cells obtained from the animal.
• Sections were mounted on silane-coated glass slides (Scientific Device Lab, Inc., Des Plains, IL), deplasticized an d incubated in 10 mM citrate buffer, pH 7.6, in a microwave oven a t 98°C for 5 minutes. Slides were then incubated with 0.5% pepsin for 30 minutes at 37°C. Apoptotic cells were detected by th e TUNEL reaction (transferase-mediated biotin-dUTP nick en d- labeling) using Klenow terminal deoxynucleotidyl transferase (Oncor, Gaithersburg, MD) in sections counterstained with 1% methyl green.
• TUNEL reaction transferase-mediated biotin-dUTP nick en d- labeling
• Klenow terminal deoxynucleotidyl transferase Oncor, Gaithersburg, MD
• TUNEL reaction was noted within cell nuclei and the cells whose nuclei were clearly brown from th e peroxidase-labeled anti-digoxigenin antibody instead of the blue- green from the methyl green were interpreted as positive.
• Plastic-embedded sections of weaned rat mammary tissue w ere used as a positive control.
• Negative controls were made b y omitting the transferase. Morphological changes characteristic of apoptosis were examined carefully to minimize ambiguity regarding the interpretation of results. With these precautions, TUNEL has been unequivocally associated with apoptosis (19).
• TUNEL has been used with DNA fragmentation an d immunohistochemical studies to demonstrate apoptosis of osteoblastic cells and osteoblasts both in vitro and in vivo (8,20). Apoptosis was also assessed in transiliac bone biopsy specimens taken from two patients with glucocorticoid-induced osteoporosis (22- and 36-yr-old, receiving 15 to 25 mg/d of prednisone for 3 to 6 yr) and from 12 age-, sex- and race-matched controls ( 13) . Two longitudinal sections were examined from each patient an d control subject. Osteoblasts were identified as cuboidal cells lining the osteoid-covered trabecular perimeter (7,9,13). Osteocytes were identified inside lacunae in mineralized bone.
• mice implanted with the higher dose of prednisolone global and spinal BMD at 27 days were significantly lower than those found in the mice that were implanted with placebo pellets (TABLE I).
• the decrease in global bone mineral density was dose dependent (P ⁇ 0.05).
• Demonstrating th e expected propensity for the axial skeleton, glucocorticoid-induced loss of bone mineral density was less conspicuous at th e hindquarters.
• the levels of serum osteocalcin, a marker of osteoblast activity were decreased more than 50% when compared to placebo, while urinary deoxypyridinoline excretion was not significantly different between the groups (TABLE I) .
• These effects were not due to changes in food intake, body weight or androgen status (TABLE II).
• hepatic fatty infiltration was absent.
• BMP Bone Mineral Density
• osteoid area decreased b y
• Bone formation rate/Bone perimeter ( ⁇ m2/(m/d) 0.15 ⁇ 0.020.13 ⁇ 0.04 0.07 ⁇ 0.03t Bone turnover (%/d) 0.68 ⁇ 0.09 0.46 + 0.12* 0.24 ⁇ O. l l t
• bone histomorphometry from these two patients showed the changes expected with chronic glucocorticoid therapy (5): reduced cancellous bone area (11.1 and 8.8%, normal is 22.4 ⁇ 1.2 SEM), decreased trabecular width (62 and 118 ⁇ m, normal is 161 ⁇ 9), decreased osteoblast perimeter (2.1 and 2.3%, normal is 7.6 ⁇ 0.4), decreased osteoclast perimeter (0 and 0.4%, normal is 0.9 ⁇ 0.2), increased reversal perimeter (13.5 and 15.4%, normal is 6.9 ⁇ 0.7) and diminished bone formation rate (0.02 and 0.05 ⁇ m 2 / ⁇ m/d, normal is 0.095 ⁇ 0.012).
• mice were chosen for these studies based on its validity as a model of the bone loss associated with loss of sex steroids (9,22) and with senescence (7), but the mouse also h as several advantages over other animals (TABLE IV).
• glucocorticoid administration consistently induces axial, greater than appendicular, bone loss without weight loss o r hypogonadism, accompanied by histological indices of impaired osteoblast function, thus reproducing the major features of th e human disease (2-5).
• the data herein also bear on recent ideas concerning the relationships between early osteoblast and osteoclast progenitors in the bone marrow.
• mature osteoclasts and osteoblasts are needed successively at each bone surface site that is being remodeled, these cells are needed simultaneously as th e basic multicellular unit (which is the instrument of bone remodeling) progresses through or across the surface of bone (41 ).
• the necessary parallel production of executive cells is accomplished by signals that originate from early members of the stromal cell-osteoblast family, which support in various ways th e production of mononuclear preosteoclasts in the bone marrow (42).
• osteoblasts become osteocytes and some become lining cells, but these fates combined do not account for all th e osteoblasts initially present. Although migration along or aw ay from the bone surface is possible, death has always seemed th e most likely alternative fate (43). Osteoblasts in remodeling bone undergo apoptosis with a frequency sufficient to account for most or all of those missing (8). Based on the dynamic histomorphometry at the murine vertebral secondary spongiosa and a wall width of about 15 ⁇ m (7,9, 14), the mean active life span of an osteoblast was calculated on cancellous bone b y dividing wall width by the mineral appositional rate.
• the mean active lifespan of a murine osteoblast is about 12 days or 288 hours.
• the prevalence of osteoblast apoptosis in the present study was 0.0066 in the placebo group.
• osteoblasts showing features of apoptosis in glucocorticoid-treated mice and human subjects could indicate no more than prolongation of the time needed for completion of the process, but it is more likely th at glucocorticoids induce apoptosis, either prematurely in cells already destined for this fate or in cells otherwise destined to become lining cells or osteocytes. In either case, the mean active lifespan of osteoblasts would be shortened and less bone formed. Thus, the reduction in bone formation by glucocorticoids could b e due to increased death as well as decreased birth of osteoblasts.
• Osteocytes are long-lived but not immortal cells. I n human rib cortical bone, their lifespan has been estimated a t about 20 years (47); if bone remains unremodeled for a longer time, the osteocytes die, as revealed by empty lacunae an d hypermineralized perilacunar bone, referred to as micropetrosis (48). Osteocyte death in cancellous bone, indicated by absence of lactic dehydrogenase activity, increases in prevalence with age in the upper femur but not in the vertebrae (49), probably because of the higher bone turnover in the spine. Empty lacunae an d enzyme absence can reveal the fact, but not the mode, of death .
• Osteocyte apoptosis has recently been detected in human iliac cancellous bone and its prevalence was increased b y pharmacological induction of estrogen deficiency ( 19).
• the present invention demonstrated that chronic glucocorticoid administration, both to mice and to human patients, likewise increases the prevalence of osteocyte apoptosis.
• the proportion of apoptotic osteocytes was much higher than of osteoblasts, reflecting the unique unavailability of osteocytes for phagocytosis because of their anatomic isolation from scavenger cells, and th e need for extensive degradation to small molecules to dispose of the cells through the narrow canaliculi. As a result, the process is prolonged and affected cells accumulate.
• the network of osteocytes probably participates in th e detection of microdamage and the transmission of signals that lead to its repair by remodeling (50). Disruption of the network b y osteocyte apoptosis could compromise this mechanism, leading to microdamage accumulation and increased bone fragility (51 ).
• Glucocorticoid- induced osteocyte apoptosis a cumulative and unrepairable defect, would explain the correlation between total dose an d incidence of avascular necrosis of bone (53) and its occurrence after glucocorticoid administration had ceased.
• the present invention has demonstrated that the mouse is a valid and informative model of glucocorticoid- induced bone disease, not confounded by weight loss or sex- steroid deficiency, and that many of the effects of chronic glucocorticoid administration on bone can be explained b y decreased birth of osteoblast and osteoclast precursors an d increased apoptosis of mature osteoblasts and osteocytes.
• Glucocorticoids inhibit bone resorption and promote apoptosis in rat osteoclasts in vitro (31), whereas bone resorption is stimulated in neonatal mouse calvaria (32).
• Glucocorticoids stimulate bone nodule formation from rat calvarial cells in vitro (33) but inhibit differentiation in a murine osteoblastic cell line (34).

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• 1999
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