WO2004093995A2 - Utilisation d'antioxydants pour traiter les troubles associes a la perte osseuse - Google Patents

Utilisation d'antioxydants pour traiter les troubles associes a la perte osseuse Download PDF

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WO2004093995A2
WO2004093995A2 PCT/GB2004/001695 GB2004001695W WO2004093995A2 WO 2004093995 A2 WO2004093995 A2 WO 2004093995A2 GB 2004001695 W GB2004001695 W GB 2004001695W WO 2004093995 A2 WO2004093995 A2 WO 2004093995A2
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bone
glutathione
levels
agent
antioxidant
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WO2004093995A3 (fr
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Timothy John Chambers
Karen Fuller
Christopher John Jagger
Jenny Maree Lean
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St George's Enterprises Limited
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Definitions

  • the present invention relates to methods of treating or preventing disorders associated with bone loss, such as osteoporosis. More particularly, the mvention relates to the use of agents that directly or indirectly increase oxidant defences, e.g. antioxidants, in the treatment of such disorders.
  • agents that directly or indirectly increase oxidant defences e.g. antioxidants
  • Multinucleated osteoclasts are specialised cells in bone that resorb mineralised bone matrix. This function is performed by secreting acid to dissolve the mineral matrix and secreting proteases to degrade exposed matrix proteins. It has been shown in genetically altered mice that a TNF (tumour necrosis factor) family member RANKL (receptor activator of nuclear factor- ⁇ B ligand) is required for activation of the osteoclast cell lineage, and preliminary attempts to target this pathway to affect bone loss have been made (reviewed in Goltzman, Nature Reviews - Drug Discovery, 2002(1), p784-795).
  • TNF tumor necrosis factor
  • RANKL receptor activator of nuclear factor- ⁇ B ligand
  • osteoblasts are responsible for bone formation. These cells secrete type I collagen and other bone matrix components. Minerals, growth factors and enzymes are also released into the matrix. The regulation of osteoblasts is thus important in maintaining bone density.
  • Parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) are important regulators, and act to promote proliferation and differentiation, and inhibit apoptosis of osteoblasts.
  • Other growth factors such as insulin-like growth factor 1 also act to stimulate osteoblast function.
  • Localised bone loss is usually associated with localised lesions e.g. caused by neoplasm or infection. Localised bone loss can also be caused by inflammation in or near bone, as occurs for example adjacent to inflamed synovium in rheumatoid arthritis, or in periodontitis. Localised bone loss may also cause tooth loss.
  • Osteoporosis is a common disorder of bone. Individuals affected by this disorder have qualitatively normal bone, but the bone is present in reduced quantities. The normal balance of bone resorption and generation is disturbed, and resorption predominates.
  • the disease may be generalised, regional or localised, and may be found alone or associated with a number of disease states. For example, generalised osteoporosis is normally age related. The incidence of osteoporosis increases with age in both men and women. A particularly high incidence of the disease is found in postmenopausal women.
  • Osteoporosis may also result from or be found in parallel with endocrine disorders such as Cushings disease (caused by excessive levels of adrenocorticotropin), acromegaly (excessive secretion of growth honnone), hyperparathyroidism and hyperthyroidism. Pregnancy,T ⁇ eparin administration and alcoholism may also be associated with generalised osteoporosis.
  • Osteoporosis also occurs in patients treated with glucocorticoids, such as rheumatoid arthritis patients, and in patients who are administered immunosuppressive therapy to prevent rejection of organ transplants.
  • Rheumatoid arthritis and other chronic systemic inflammatory diseases also cause osteoporosis, independently of glucocorticoid therapy.
  • osteoporosis examples include idiopathic juvenile osteoporosis, and osteoporosis may alternatively accompany plasma cell myeloma, Gauchers disease, glycogen storage disease, anaemia, nutritional deficiencies (e.g. anorexia), diabetes mellitus, immunodeficiency and chronic liver disease.
  • idiopathic juvenile osteoporosis may alternatively accompany plasma cell myeloma, Gauchers disease, glycogen storage disease, anaemia, nutritional deficiencies (e.g. anorexia), diabetes mellitus, immunodeficiency and chronic liver disease.
  • Regional osteoporosis may be caused by disuse or immobility of the limbs, and may be reversible (such as regional migratory osteoporosis).
  • the prevalence of osteoporosis is high. In the United States for example, it has been calculated that 35% of postmenopausal white women and 19% of white men have the disease (reviewed in Riggs et al., Endocrine Rev., 2002, 23(3), p279- 302). The sufferers are more likely to incur fractures and these can pose serious threats to the life of the sufferer. With the increases in life expectancy that are occurring worldwide, the prevalence of this disease is likely to increase.
  • Estrogen has a variety of roles in the skeleton, and acts at a number of different levels. At the organ level, estrogen has a very important role in acting to conserve bone mass. At the tissue level, estrogen suppresses bone turnover to maintain balanced rates of bone formation and loss, and at the cellular level, the hormone affects a number of properties of osteoblasts and osteoclasts (e.g. it decreases osteoclast formation and activity). In line with these observations, osteoblasts and osteoclasts contain functional estrogen receptors, of both the and ⁇ type. The molecular mechanisms by which these estrogen hormones act in bone were not clear prior to this invention. Cytokines such as TNF and RANKL or decoy receptors such as OPG were however postulated as being involved in increased bone resorption, while increased apoptosis was thought to underlie deficient bone fo ⁇ nation.
  • Cytokines such as TNF and RANKL or decoy receptors such as OPG were however postulated as being involved in increased bone resorption,
  • Hormone replacement therapy which involves the administration of oral forms of 17 ⁇ estradiol, estrone and conjugated equine estrogens, in addition to transdermal estrogens, is a widely used treatment for bone loss, particularly osteoporosis.
  • This approach has the drawback that administration of estrogen must also be accompanied by administration of progestin if the patient has not undergone a hysterectomy.
  • a further drawback is the associated risk of coronary heart disease, breast cancer, stroke and pulmonary embolism.
  • Estrogen derivatives such as 17 , ⁇ 8,9-dehydroestradiol, 17 ⁇ , ⁇ 8,9- dehydroestradiol and estra-5(10),7-dienes (such as estra-5(10),7-dien-3 ⁇ -ol-17-ones, or its 3 sulphate ester, or estra-5(10),7 dien-3 ⁇ -ol-17-one 3 glucuronides, estra- 5(10),7-dien-3 ⁇ -ol-17-ones, or its 3 sulphate ester, or estra-5(10),7-dien-3 ⁇ -ol-17- one 3 glucuronides) have estrogenic activity in that they promote mouse uterine growth and have also been proposed as useful in providing estrogen replacement therapy and hence also as a treatment for osteoporosis.
  • SERMs selective estrogen receptor modulators
  • Goltzman, 2002, supra It has been suggested that these agents primarily enhance the quality rather than the quantity of bone, as assessed by the relatively low associated increases in bone mass density. These agents however present an increased risk of thromboembolism, despite not having all of the disadvantages of estrogen treatment such as increased risk of uterine cancer.
  • SERMs act on the ER (estrogen receptor) in bone cells and are thus used to treat osteoporosis.
  • Bone mass density is also increased in clinical treatment by bisphosphonates, such as alendronate and risedronate. These compounds have also been shown to reduce the incidence of vertebral and hip fractures.
  • bisphosphonates unlike estrogens and SERMs, can be applied to a wide variety of skeletal disorders such as Paget's disease, hypercalcaemia of malignancy, metastatic bone disease, multiple myeloma and osteogenesis imperfecta.
  • bisphosphonates are laid down in bone, where they remain for very long periods, and the long-term consequences of this are unknown.
  • Calcitonin has also been shown in some but not all studies to be effective in the treatment of osteoporosis, Paget's disease and hypercalcaemia of malignancy either by injection or nasal inhalation.
  • Anabolic agents that act to stimulate the formation of new bone have also been used in the treatment of bone loss disorders.
  • Fluoride represents one example of such anabolic agents.
  • the new bone produced is of poor quality and this leads to an increased number of fractures, and increased bone pain.
  • BSO buthionine sulphoximide
  • the glutathione system is a major regulator of cell signalling and acts as an antioxidant system to scavenge free radicals and forms part of the cells' oxidant defences in response to oxidative stress such as is caused by reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • the levels may however fluctuate and if ROS levels increase, this is detected by the cell and leads to induction of various enzymes such as gamma glutamylcysteine synthase, catalase and superoxidase dismutase (SOD). . . "
  • Glutathione is a tripeptide that is synthesised by cells via the enzymes gamma glutamylcysteine synthase and glutathione synthetase. It is present in cells at high concentrations, sufficient to act as a ROS scavenger. It possesses a reduced sulphydryl group and is oxidised to "oxidised glutathione" (GSSG) by oxidising agents. The oxidised glutathione is then reduced back to "reduced glutathione" (GSH) by glutathione reductase. Glutathione may be found in oxidised or reduced form in bone tissue. The reduced form makes up >95% of total glutathione.
  • GSH also participates in oxidant defences by acting as an electron donor for enzymatic removal of ROS.
  • glutathione peroxidase enables the removal of hydrogen peroxide, a ROS that is generated by many living processes, both as an inevitable consequence of processes such as mitochondrial electron transport and in a cytokine-regulated manner by enzymes such as NADPH oxidases.
  • thioredoxin a polypeptide that acts on target proteins to restore the reduced state of thiols. Analogous to glutathione, oxidised thioredoxin is converted back to the reduced fo ⁇ n by thioredoxin reductase.
  • thioredoxin is a cofactor in many cell signalling interactions, and represents an important mechanism whereby cell signalling is modulated by the redox state of the cell. For example, it controls the activity of a number of transcription factors.
  • the redox state of the cell is determined by the ratio of reduced to oxidised glutathione. There is much evidence that cell signalling is modulated by the redox state of the cell. Thus, glutathione can act not only as an oxidant defence molecule, but can modulate cell signalling and thereby control cell function.
  • ROS such as hydrogen peroxide
  • ROS are highly diffusible and relatively stable.
  • Hydrogen peroxide acts as a signalling molecule in cells.
  • glutathione- peroxidase catalysed oxidation of GSH glutathione- peroxidase catalysed oxidation of GSH. Therefore, lower levels of glutathione or glutathione peroxidase can cause an increase in the concentration of hydrogen peroxide in tissues.
  • Vitamin C administered at levels of 2 mmole/kg/day mi ⁇ ored the effect of administration of estrogen on the level of glutathione. Vitamin C at this high level was also able to prevent the effects of ovariectomy on bone loss. Furthermore, N-acetyl cysteine, which also increases glutathione levels, similarly prevented bone loss in ovariectomised mice.
  • vitamin C Whilst the potential link between vitamin C and bone disease has been investigated, the vitamin was not used at oxidant defence-inducing levels. Furthermore, vitamin C was examined in view of the importance of this factor in the disease scurvy and in the formation of collagen. The benefit of its " potential antioxidant properties were not appreciated. For example, Bjarnason et al. (Osteoporosis Int., 2001, 12(15), p380-4) tested the effects of vitamin C (500 mg per day) on markers of bone loss (serum osteocalcin and total alkaline phosphatase) in 68 elderly women with osteoporosis, and no significant effects were seen. The present inventors have found that antioxidant benefits are only confe ⁇ ed at higher concentrations than those used in these experiments.
  • Isoflavones which have weak antioxidant properties, have also been tested for their effects on bone density in postmenopausal women as they also have weak estrogenic activities (Cecchini et al, Calcif. Tissue Int., 1997, 61, p59-ll; Hsu et al., J. Reprod. Med., 2001, 46(3), p221-6). Neither of these studies were conclusive.
  • the present invention provides a method of treating or preventing bone loss disorders in a human or non-human animal, comprising the step of administering an agent which increases the levels of oxidant defences and/or at least one antioxidant in said animal, preferably in bone cells in said animal.
  • the agent is administered at a dose which increases the levels of said oxidant defences and/or at least one antioxidant to therapeutically effective levels.
  • the present invention further provides: use of an agent which increases the levels of oxidant defences and/or at least one antioxidant, preferably in a cell, in the manufacture of a medicament for treating or preventing a bone loss disorder in a human or non-human animal; a method of screening for agents suitable for use in treating or preventing a bone loss disorder, which method comprises:
  • (c) determining whether or not the candidate agent may be suitable for said use; and a method of diagnosing or monitoring a bone loss disorder in a human or non-human animal, which method comprises determining the level of oxidant defence and/or one or more antioxidants in a sample taken from the human or non-human animal.
  • FIG 1 shows the levels of various components of the glutathione system in rat bone ma ⁇ ow following ovariectomy or sham treatment.
  • Ovariectomised rats ovx
  • ovx Ovariectomised rats
  • ⁇ E 2 17- ⁇ -estradiol
  • ⁇ E 2 17- ⁇ -estradiol
  • Figure 2 shows the effect of NAC on ovariectomy-induced bone loss in mice.
  • (a) shows bone loss in sections of femora of mice after sham treatment or ovariectomy in the presence or absence of N-acetylcysteine (NAC).
  • (b-g) show indices of bone resorption and bone formation.
  • OC osteoclasts
  • ES/BS percentage eroded surface of the bone
  • Ob osteoblasts. *p ⁇ 0.01 vs ovariectomy (Student's t-test). Data expressed as mean ⁇ SEM
  • Figure 3 shows bone loss parameters in response to ascorbate treatment,
  • (a) shows femoral sections from sham or ovariectomised mice untreated or treated with ascorbate.
  • (b-g) show parameters of bone resorption and formation as described in Figure 2;
  • Figure 4 shows the effect of l-buthionine-(S,R)-sulphoximine (BSO) on mouse bone
  • (a) shows femoral sections from a control and BSO treated mouse
  • (c- k) show the effect of BSO administration on bone loss and formation parameters.
  • n 6 mice per group. Data expressed as mean ⁇ SEM.
  • Figure 5 shows the effect of estrogen on the osteoclastic thiol antioxidant system
  • (a and b) show the effects of estrogen (E 2 ) on glutathione reductase (GR), thioreductase (TrXR) and glutathione, in osteoclasts.
  • (c) shows the effect of BSO, NAC and H 2 O 2 on TRAP-positive multinucleate cell formation
  • (d) shows the results of an electrophoretic mobility shift assay showing the effect of BSO and NAC on NFKB activity in osteoclasts in vitro.
  • M M-CSF
  • RL RANKL
  • S unlabelled self probe
  • H mutant species
  • (e) shows the effect of estrogen on TNF ⁇ RNA in in vitro osteoclasts (* p ⁇ 0.05 vs control group).
  • Figure 6 shows the ability of ascorbate to restore mouse bone volume following ovariectomy.
  • the present invention is concerned with treating or preventing bone loss disorders in a human or non-human animal by administering an agent which increases the levels of oxidant defences and/or at least one antioxidant in said animal, preferably in bone cells of the animal.
  • the agent may be an antioxidant.
  • treating refers to the reduction or alleviation, preferably to at least normal levels, of one or more of the symptoms or signs of said bone loss disorder e.g. bone mass volume or density.
  • said bone mass volume or density in a treated individual may be improved relative to an untreated individual and/or relative to the volume or density at the time of starting said treatment.
  • the bone mass volume or density may be increased for example 10, 20, 30% or more over a treatment period of e.g. several weeks to months or even years, e.g. 3-6 months.
  • bone mass volume or density may be improved to approach or even exceed normal levels, e.g. for example up to 60, 70, 80%) or higher of normal levels.
  • treatment refers to reducing the extent of bone loss as well as to the reversal of bone loss, i.e. the restoration of bone that has been lost.
  • the invention provides a method of treating bone loss as described hereinbefore wherein bone mass volume or density is increased as a consequence of said treatment.
  • Levels of bone volume or density are determined by standard means known in the art including radiographic imaging, single or dual photon absorptiometry, X-ray absorptiometry, ultrasound densiometry and quantitative CT.
  • Preventing refers to absolute prevention i.e. the absence of detectable changes in bone loss, e.g. bone mass density, or the reduction or alleviation of the extent or timing (e.g. delaying) of the onset of that bone loss. This term also refers to the prevention of further bone loss, for example after treatment which may have achieved maintenance of bone density or improved bone density or volume.
  • a "bone loss disorder” is any condition wherein the bone mass density and/or volume is reduced. Such disorders include those in which bone loss is a primary or secondary symptom of said disorder. Disorders include diseases with an underlying pathological disturbance relative to a normal individual which may result for example from infection or an acquired or genetic imperfection. Disorders also include those resulting from intentional or unintentional ingestion of certain agents, e.g nutritionally associated conditions or alcoholism. Disorders however also include conditions generally considered to be normal, e.g. through the ageing process, or conditions attributable to physiological or surgical events, e.g. menopause, pregnancy or ovariectomy, respectively, but which exhibit undesirable bone loss.
  • the disorder is selected from the group consisting of rheumatoid arthritis, periodontitis, osteoporosis, Paget's disease, hypercalcaemia of malignancy, metastatic bone disease, multiple myeloma, osteogenesis imperfecta, osteomalacia, hyperparathyroidism and hypoparathyroidism.
  • the condition is osteoporosis, especially preferably osteoporosis resulting from estrogen deficiency.
  • Animals which may be subject to methods of the invention are preferably mammalian, preferably humans and companion, laboratory or agricultural animals such as dogs, cats, monkeys, horses, sheep, goats, cows, rabbits, rats and mice. Especially preferably however the methods of the invention are applied to humans.
  • antioxidant refers to any entity that delays or prevents the process of oxidation, or can counteract the damaging effect of oxygen or any free radical with an unpaired electron, or a reactive oxygen species.
  • antioxidants are free radical scavengers (scavenger antioxidants).
  • antioxidants may occur naturally, e.g. glutathione or ascorbic acid or may be antioxidants not normally present in a cell, e.g. lipoic acid; Oltipraz; Trolox.
  • Antioxidants are considered to be those entities which satisfy standard pharmacological tests which examine antioxidant properties.
  • an agent is considered to be an antioxidant if it satisfies the in vitro test described in WO98/16544, e.g. if the antioxidant is capable of inhibiting LDL oxidation, e.g. more than 50%, preferably more than 75% under the conditions described in that document.
  • antioxidant levels are those which are therapeutically effective in preventing and/or treating bone loss. Since the addition of antioxidants increases the pool of available antioxidants, it is believed that this increase can in effect influence the quantity of reduced glutathione, or the ratio of reduced to oxidised glutathione. Preferably the level of antioxidant which is used in methods of the invention is sufficient to achieve increases in this quantity or ratio as described hereinafter.
  • Oxidant defences refers to the sum of all the defences against such oxidants. Such defences are activated by the cell in response to the presence of an oxidant or to conditions of oxidative stress. Oxidant defences include:
  • antioxidant scavengers such as glutathione, alpha tocopherol, carotenoids, bilirubin and ascorbate;
  • antioxidants e.g. enzymes or cofactors involved in the production or uptake or secretion or utilisation of antioxidants, such as glutathione peroxidase, gamma glutamylcysteine synthetase, glutathione reductase, thioredoxin, thioredoxin reductase
  • other enzymes that act to remove ROS with or without the help of other enzymes (e.g. superoxide dismutases, catalase, peroxidases); and
  • proteins that minimize pro-oxidant availability e.g. by sequesting transition elements which would otherwise generate ROS from a variety of cell constituents (e.g. transferrins, haptoglobulins, metallothionine and caeruloplasmin).
  • Increases in oxidant defences may be identified by examination of the levels of particular components, e.g. as described above, or more generally by examining the levels of specific species which reflect the levels of oxidant defences which are present. Thus for example, increases in total and/or reduced glutathione may be examined, wherein increases are indicative of increased oxidant defences. When this measure is used preferably said reduced and/or total glutathione content increases at least 5% relative to the levels in the individual to be treated, e.g. at least 10 or 20%.
  • reduced glutathione may make up at least 95%, preferably at least 96, 97, 98 or 99%) of the total glutathione content, e.g. in a cell under examination.
  • levels are increased to levels at which bone loss is prevented, curbed or reversed in vivo as described hereinbefore. Conveniently, this may be assessed using the indices of bone resorption and/or bone formation described in the Examples.
  • the levels are increased so that they more closely approximate those in normal individuals. For example for therapy of postmenopausal bone loss, the levels are increased so that they more closely approximate those in premenopausal or young, normal individuals or are e.g. within 10 or 20%o of such levels, and preferably achieve such levels.
  • the agent induces or increases antioxidant levels and/or oxidant defences in a cell, tissue, organ or mammal, when compared to the antioxidant levels and/or oxidant defences in a cell, tissue, organ or mammal in the absence of the agent or oxidant defence component.
  • This is preferably achieved by increasing the level of at least one antioxidant and/or oxidant defence component.
  • the oxidant defences and/or antioxidant capacity of the cell, tissue, organ or mammal is thus influenced by administration of the agent.
  • one of the goals of the invention is to increase the tissue content of endogenous antioxidants (e.g. glutathione) in the reduced form.
  • endogenous antioxidants e.g. glutathione
  • glutathione and thioredoxin play a pivotal role in cell defence systems acting as antioxidants as well as cofactors for the enzymatic destruction of ROS and act as sensors for the presence of ROS (e.g. thioredoxin acts as a signalling cofactor, but only when in its reduced form).
  • one of the purposes of the agents described herein is therefore to increase the concentration of the total and/or reduced form of these molecules and thus allow these molecules to perform their unique roles within the cell.
  • This may conveniently be achieved by alleviating their role as antioxidants, which can be performed by other less specialized antioxidants, e.g. radical scavengers.
  • oxidant defences may be increased by the addition for example of high levels of antioxidants (scavenger antioxidants), preferably exogenous antioxidants.
  • scavenger antioxidants When scavenger antioxidants are present, they prevent oxidative damage, e.g. by acting as a free radical scavenger.
  • Their action to remove ROS effectively spares reduced glutathione in that less reduced glutathione is required to remove the ROS.
  • the removal of ROS results in relatively more of the reduced form being present as it is no longer involved in the removal of ROS.
  • ROS affect cell signalling, e.g. by activation of NFKB.
  • the effects of ROS on signalling are seen at relatively low levels of ROS and are mainly controlled by reduced glutathione and thioredoxin and may therefore be lowered by the use of molecules which allow maintenance (or restoration) of glutathione and/or thioredoxin in the reduced form.
  • Oxidant defences may however also be improved by techniques which do not necessarily increase the level of antioxidants, e.g. by decreasing the levels of oxidants (e.g. by SOD/catalase mimetics such as EUK 134) and all such methods are encompassed in methods of the invention.
  • an "agent” that increases the levels of antioxidants and/or oxidant defences, e.g. in tissues, may cause this increase directly or indirectly.
  • the agent may be taken up by the cells of the body or may be present in interstitial or body fluids.
  • methods of the invention to treat or prevent bone loss by administering an agent which increases the levels of at least one antioxidant consists of administration of an antioxidant and the level which is increased relates to the level of the antioxidant which is administered.
  • Agents that stimulate, i.e. increase the level of, antioxidant defences may suppress the potent osteoclast- forming factor trx, which is induced in response to oxidative stress.
  • Agents that "directly" increase antioxidants levels are molecules that are themselves antioxidants and on administration, e.g. to the cell, exhibit antioxidant behaviour, e.g. in or around that cell or other relevant cells e.g. bone cells.
  • Agents that "directly" increase oxidant defences are themselves components of the oxidant defence system, e.g. catalase, thioredoxin reductase and glutathione reductase or functionally equivalent fragments, variants or analogues thereof.
  • Agents that "indirectly" increase antioxidant levels in a cell may be precursors or comprise subunits of an antioxidant which is formed by processes which occur in the cell. Alternatively such agents may influence the levels of one or more antioxidants within a cell through manipulation of cellular processes controlling the same, e.g. by providing or affecting (e.g. activating) enzymes that produce antioxidants.
  • Agents that "indirectly" increase oxidant defences are agents that ultimately result in alterations in the level of oxidant defences, in which that alteration is achieved via the intermediacy of one or more other molecules, e.g. as described in respect of agents that indirectly increase antioxidant levels.
  • antioxidant levels and/or oxidant defences are increased by activating the glutathione/ thioredoxin oxidant defence system (which is an important defence mechanism in bone ma ⁇ ow).
  • said agent which increases antioxidant levels does so by activating the glutathione/thioredoxin oxidant defence system.
  • Activation of this system may be assessed by examining the total content of particular components of the glutathione/thioredoxin pathway in the cell, preferably in bone marrow cells. For example, the total glutathione levels may be examined. Alternatively the levels of a particular component may be examined, e.g. glutathione reductase activity or levels, gamma glutamylcysteine synthase activity or levels, thioredoxin reductase activity or levels, or thioredoxin activity or levels may be examined. Such measurements provide an indication of the antioxidant or oxidant defence levels which are present.
  • Glutathione levels may be measured by for example using the test described in the Examples.
  • the levels of other components in the pathway may be determined by any appropriate means, including those described in the Examples.
  • Agents which affect the glutathione/thioredoxin defence pathway include enzyme activators or the enzymes themselves or any other agents that stimulate the pathway.
  • the enzymes which are targeted are glutathione reductase, gamma glutamylcysteine synthase, thioredoxin reductase, glutathione peroxidase or thioredoxin.
  • the agent may thus be, for example, an enzyme which may be administered in the proteinaceous form or the nucleic acid molecule encoding the same may be used to transfect the cells.
  • the agent which is used causes a rise in endogenous antioxidants in bone cells, e.g. glutathione or thioredoxin, " or in oxidant defences in bone cells.
  • Appropriate agents for use in the invention may be identified in vitro. Methods of screening for such agents, the agents thus identified and their therapeutic uses described herein form further aspects of the invention. Conveniently this may be achieved by exposing cells, especially osteoblasts, osteoclasts or other types of cells in bone and bone marrow to candidate agents.
  • the ability of the agent to increase the level of one or more of the components of the oxidant defence system, such as (but not exclusively) glutathione, thioredoxin, or their reductases can be assessed as described in the Examples.
  • such agents can be identified via the induction of changes in expression of RNA, or by Western blot, or by using a reporter molecule under the control of the gene for the appropriate enzyme.
  • Agents may be tested in vivo for their ability to increase one or more components of the oxidant defences. As an example levels of glutathione, thioredoxin and their reductases may be measured, as described in the Examples. Agents may also be tested directly for their ability to prevent bone loss as described in the Examples. Agents, such as antioxidants, that suppress trx expression in osteoclasts and their precursors may be identified.
  • Agents suitable for use in the invention are diverse and as mentioned above include all antioxidants, and all agents that enhance oxidant defences. They may be naturally or endogenously occurring or may be synthetic or exogenous. The following non-exhaustive list, includes examples of agents that may be used or which may be used to derive appropriate agents: adenosine;
  • AEOL11201 (Choudhary et al, 2001, Dig. Dis. Sci., 46(10), p.2222-30); AGI-1067 (US Patent No. 6147250); aminosalicyates (alone or as components, of sulphasalazine); 21-aminosteroids (e.g. PNU-74389G - see Rauscher et al., 2000, J. Biochem. Mol. Toxicol., 14(4), p.l89-194; angiotensin converting enzyme inhibitors including captopril and enalapril; apomorphine; ascorbate (vitamin C); astaxanthin; azulenzyl nitrones and derivatives, e.g. stilbazulenyl nitrone; benzopyran analogues, KR-31378 (see Hong et al., 2002, J. Pharmacol. Exp. Ther., 301(1), p.210-216);
  • BO-653 (Inoue et al, 2002, Artherosclerosis, 161(2), p.353-363); butylated hydroxyanisole (BHA); butylated hydroxytoluene (BHT); carotenoids such as lycopene, ⁇ -carotene, ⁇ -carotene, ⁇ -cryptoxanthin, zeaxanthin, lutein, echinenone, canthaxanthin and astaxanthin; carnosine and derivatives such as homocarnosine and anserine; carvedilol and metabolites, e.g. SB211475 and other ⁇ -blockers; chlorpromazine; cimetidine;
  • Coenzyme Q(10) and derivatives including idebenone; CPI-1189 (Clifford et al., 2002, Neurology, 59(10), p.1568-1573); curcumin and analogues, especially bis ⁇ l,7-(2-hydroxy-phenyl)-hepta-l,6-diene-3, 5- dione; cysteine and derivatives or precursors such as letosteine, N-acetylcysteine, procysteine, N,N'-diacetyl-L-cystine (DiNAC), bucillamine, S-adenosyl-L- methionine, cysteamine, L-2-oxo-4-thiazolidine- carboxylate, methionine, glutathione methyl ester and S-allylcysteine;
  • cysteine and derivatives or precursors such as letosteine, N-acetylcysteine, procysteine, N,N'-di
  • Daflon-500 (a combination of 90%> diosmin and 10% hesperidin marketed by Servier as S-5682); desferrioxamine, and other natural iron chelators; diaminouracil derivatives including CX-659S (see Goto et al., 2002, Eur. J.
  • DPPD diphenylphenylenediamine
  • ADT anethole dithio
  • Sulfarlem from Solvay Pharma
  • Oltipraz from Aventis dobesilate; ebselen and other organic selenium antioxidants; enaminones, especially 3-substituted amino- l-aryl-6-hydroxy-hex-2-ene-l -ones; entecapone; enzymes such as CuZnSOD, MnSOD or EC-SOD; enzyme activators or mimics such as 4-phenyl butyrate (inducer of superoxide dismutase) and EUK 134, 8 or 189
  • Desferal-Mn(IV) and related Mn complexes, copper complexes and TMPyP and other metalloporphyrins e.g. AEOL 10113 and AEOL 10150, see Habeck, 2002,
  • flavones such as 4-bromoflavone, 4-chloroflavone, 4-trifluoromethylflavone, 3- bromoflavone and 3-chloroflavone
  • fullerenol (polyhydroxylated C(60)) derivatives such as hexasulfobutylated C60
  • FC 4 S furanone derivatives, especially 4,5-diaryl-3-hydroxy-2(5H)furanones; gamma glutamylcysteine ethyl ester; galalate (e.g. propyl galalate); ginkgo biloba and extracts (e.g. EG 6761 ginkgolide B and bilobalide) and mimics
  • NV-31 see Ahlemeyer et al., 2001, Brain Res., 890(2), p.338-42
  • gliclazide e.g. glutathione and its derivatives and precursors such as gamma glutamylcysteine ethyl ester; glutathione ethyl ester, 1-152 (see Oiry et al, 2001, Bioorg. Med. Chem.,
  • 6-hydroxy- 1 ,4-dimethyl-carbazole 6-hydroxy- 1 ,4-dimethyl-carbazole; indoles containing a triazole moiety; isoeugenol; isothiocyanates such as methylsulfinylalkyl isothiocyanates, such as sulforaphanes
  • aromatic isothiocyanates including ⁇ - phenylethylisothiocyanate (PEITC) and benzyl isothiocyanate; and 7- methylsulfmylheptyl- and 8-methylsulfinyloctyl-isothiocyanates (Rose et al,
  • LY178002 LY256548; lycopene; mangiferin and other xanthine glucosides;
  • MCI-186 see Ninomiya et al., 2002, Transplantation, 74(10), p.1470- 1472; melatonin and melatonin receptor ligands such as N-[(4-methoxy-lH-indol-2- yl)methyl]propanamide;
  • N N-diphenyl-p-phenylene diamine nitecapone
  • nitrile crambene ⁇ utr. Cancer 42, 233-240, 2002
  • non-calcineurin-binding analogues of FK506, especially GPI 1046 and V10367 see
  • OPC-14117 a compound with structural homology to vitamin E, and related compounds (see Aoyama et al., 2002, Brain Res., 934(2), p.l 17-124); oxerutin;
  • Oxigon indole-3-propionic acid
  • penicillamine pentoxifylline
  • phenylbutazone plant phenolics (such as anthocyanidins, e.g. malvidin, cyanidin and apigenidin; phenylpropanoids such as caffeic acid, ?-coumaric acid and chlorogenic acid; aurones; tocopherols; chalcones; thymol; vanillin; eugenol; hydroxytryosol; camosic acid; carnosol; guaiacol; rosmaric acid; guaiacol; dehydrozingerone; nordihydroguaiaretic acid; pelargonidin; sesamol; cathechins such as epicatechin and epigallocatechin-3-gallate; galeic acid; gossypol; flavanols; flavanones; flavanoids; isoflavone glycosides such as gensitein
  • PYC Pycnogenol
  • py ⁇ olopyrimidines e.g. PNU-104067F, see Rauscher et al., 2000, J. Biochem., Mol.
  • Toxicol. 14(4), p.l89-194; resveratrol and analogues, e.g. astringinin; saralasin and other antioxidant angiotensin III receptor antagonists; selegiline; silybin-beta-cyclodextrin;
  • MDL74,405 see Tang et al, 1995, Am. Heart J., 130(5), p.940-948
  • transferrin trimetazine and derivatives such as S-15176 (see Settaf et al., 2000, Eur. J.
  • triterpenes e.g. celastrol
  • troglitazone e.g. ubiquinone
  • W-2721 and WR-1065 (amifostine), (see Grdina et al, 2002, Mil. Med., 167(2 suppl), p.51-53); xanthine oxidase inhibitors;
  • A is chosen from a
  • R 3 is chosen from a C,-C 6 alkyl group, a C,-C 6 alkyl group substituted with one or more groups chosen from hydroxyl, amino, chloro and C,-C 4 alkoxy groups, an aryl (C,-C 6 alkyl) group, a (C C 6 alkyl) carbonyl group and an aryl (C,-C 6 alkyl) carbonyl group,
  • R 4 being a C,-C 6 alkyl group or an aryl group, and a CHOH group
  • R j and R 2 are chosen, independently of one another, from hydrogen, a halogen, a nitro group, a nitroso group, a thiocyano group, a C r C 6 alkyl group, a C 2 - C 6 alkenyl group, an aryl group, an aryl(C r C 6 alkyl) group, an aryl(C 2 -C 6 alkenyl) group, a carboxyl group, a (C r C 6 alkyl)carbonyl group, an arylcarbonyl group, a (C C 6 alkoxy)carbonyl group, a (C r C 6 alkoxy)carbonyl(C r C 6 alkyl) group, a C,-C 6 alkoxy group, a trifluoromethyl group, an amino group, a di(C r C 6 alkyl)
  • R is chosen from a C C 6 alkyl group
  • Y " is a pharmaceutically acceptable anion such as halide or sulphate, and their pharmaceutically acceptable salts.
  • Particularly suitable such compounds are ones in which:
  • X is chosen from S and O,
  • A is chosen from a
  • R 3 is chosen from a C ] -C 6 alkyl group, a C,-C 6 alkyl group substituted with one more groups chosen from hydroxyl, amino, chloro and C r C 6 alkoxy groups, an aryl(C r C 6 alkyl) group, a (C r C 6 alkyl)carbonyl group and an aryl (C r C 6 alkyl) carbonyl group), a C ⁇ O group, a
  • R 4 being a C r C 6 alkyl group, and a CHOH group
  • R, and R 2 are chosen, independently of one another, from hydrogen, halogen, a C r C 6 alkyl group, an aryl group, an aryl (C r C 6 alkyl) group, a carboxyl group, an alkoxycarbonyl group, a C r C 6 alkoxy group, a trifluoromethyl group, a di(C C 6 alkyl) amino (C r C 6 alkyl) group and an acylamino group of formula -NHCOC n H 2n+I with n from 0 to 6, or alternatively R j and R 2 together from a C 2 -C 12 alkylene group, optionally with the exception of the 2,2-dimethyltrimethylene group, or a C 3 -C 12 cycloalkylene group,
  • R is chosen from a C j -C 6 alkyl group, and their pharmaceutically acceptable salts.
  • an aryl group or aryl fraction of an arylalkyl group denotes an aromatic carbon-based group such as a phenyl or naphthyl group or an aromatic heterocyclic group such as a thienyl or furyl group, it being possible for these groups to bear one or more substituents chosen from a halogen atom, a C C 4 alkoxy group, a trifluoromethyl group, a nitro group and a hydroxyl group.
  • R j is alkyl, preferably with 1-6 carbon atoms and more preferably methyl, phenyl, substituted phenyl, cyclohexenyl, or substituted cyclohexenyl;
  • R 2 is methyl, phenyl or CO 2 R 3 wherein R 3 is alkyl .such as C,-C 6 alkyl or phenyl; and, optionally, when X is O and R, is phenyl, R 2 is not methyl.
  • esters are compounds of formula (II) in which X is O, R ⁇ is unsubstituted phenyl or methyl and R 2 is CO 2 Me.
  • R j is a substituted phenyl
  • the phenyl is substituted at the 4 position with a methoxy, t-butyl, alkyl ketone, sulfonamide, or trifluoromethyl group.
  • R is a substituted cyclohexenyl
  • the cyclohexenyl is preferably substituted in the 2 position with a halogen, an alkyl, or a phenyl.
  • X is O
  • R [ is (4-Meo)phenyl, (4-t-Bu)phenyl or cyclohexenyl and R 2 is methyl;
  • X is O, R[ is phenyl, and R 2 is alkyl, and preferably R 2 is methyl, ethyl, propyl, or isopropyl.
  • Suitable agents are also disclosed in US Patent No. 5658913. These agents are pyrazine derivatives having the following formula:
  • R [ represents a hydrogen atom or a C,_ 3 alkyl group
  • the compounds (III) wherein R[ is a hydrogen atom or methyl group and R 2 is a group represented by the formula: -C(R a ) C(R b )(R a ) wherein R a , R b and R c , being the same or different from each other, means a hydrogen atom or methyl group are prefe ⁇ ed.
  • Sulforaphane analogues described in US Patent No. 5411986.
  • Sulforaphane is (-)l-isothiocyanato-(4R)-(methylsufmyl)butane.
  • the analogues have a first moiety which is an isothiocyanate, a second moiety which is a polar functional group and a chain of one or more carbon atoms linking the first and second moieties.
  • the analogue contains no pyridyl moieties.
  • Prefe ⁇ ed compounds are: exo-2-acetyl-6-isothiocyanatonorbornane (GHP 1066), exo-2-acetyl-5- isothiocyanatonorbornane (GHP 1067), exo-2-isothiocyanato-6- methylsulfonylnorbornane (GHP 1068), cis-l-isothiocyanato-4- methylsulfonylcyclohexane (GHP 1073), 6-isothiocyanato-2-hexanone (CH 3 CO(CH 2 ) 4 NCS) (GHP 1105) and 6-isothiocyanato-2-hexanor(GHP 1106).
  • radicals R 1 to R 9 are H, radicals chosen from alkyl, alkenyl, alkinyl, aryl, arylalkyl, alklaryl, heteroaryl, heteroalkyl and hetero(alkylaryl and arylalkyl), preferably having 1 to 20 carbon atoms, or chains of the formula (R 5 xR 6 ) n wherein n>l, x represents one or more heteroatoms and R 5 and R 6 are radicals chosen from alkyl, alkenyl, alkinyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroalkyl and hereto- (alkylaryl and arylalkyl) having 1 to 20 carbon atoms.
  • Alkyl is typically C,-C 6 alkyl
  • alkenyl is typically C 2 -C 6 alkenyl
  • alkinyl is typically C 2 -C 6 alkinyl
  • aryl is typically phenyl
  • the heteroatom in the hetero group is usually N, O or S and from 1 to 3 such heterotoms may be present, and n may be 1 to 4.
  • the radicals R 2 to R 9 and H and the radical R 1 is a radical of the formula (V):
  • Suitable purine derivatives for use in the invention are the purines of US Patent No. 5801159. These purines have the following formula (VI):
  • A is a sugar or a phosphorylated sugar, particularly ribose, deoxyribose, and phosphorylated derivatives thereof, particularly 3' and/or 5 '-phosphate and 5'- pyrophosphate, including derivatized phosphates, such as phosphate esters and anliydrides, particularly mixed anydrides, where the esters may be alkyl esters of alkyl groups of from 1 to 3 carbon atoms or of sugars, e.g.
  • ribose, deoxyribose and phosphorylated esters thereof, such as 3' and 5 '-esters, and the mixed anhydrides may be anliydrides with carboxylic acids, such as pyruvate, and combinations thereof, e.g. ADP-r-r-P-r-P, where r is ribosyl and P is phosphate, where the final P may be bonded to a nucleoside;
  • R and R 1 may be nitrogen, oxygen or sulfur, where the sulfur may be substituted or unsubstituted, and the remaining valences are satisfied by hydrogen, and where R may be taken with Y 1 to define a doubly bonded heteroatom;
  • Y and Y 1 are hydrogen or may be taken together to form a double bond between the atoms to which they are bonded or Y 1 may be taken together with R to form a double bond to the atom designated by R.
  • guanosine adenosine, inosine, 6-mercaptopurine riboside, adenosine diphosphate, adenosine monophosphate, inosine monoposphate, 2'-deoxyadenosine-5'-monophosphate, 2- inosine-5'-monophosphate, 2-deoxyguanosine-5'-monophosphate, S-(2-hydroxy-5- nitrobenzyl)-6-thionosine, erythro-9-(2-hydroxy-3-nonyl) adenine and S-4- mtrobenzyl-6-thioinosine.
  • a C,-C 6 alkyl group is typically a C r C 4 alkyl group or a C,-C 3 alkyl group.
  • Prefe ⁇ ed alkyl groups are methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl and tert-butyl.
  • a C r C 6 alkoxy group is typically a C C 4 alkoxy group.
  • Prefe ⁇ ed alkoxy groups are methoxy and ethoxy.
  • a C 2 -C 6 alkenyl group is typically a C 2 or C 3 alkenyl group such as allyl.
  • a C 2 -C 6 alkynyl (or alkinyl) group is typically C 2 or C 3 alkynyl (or alkinyl) group.
  • An aryl group is preferably phenyl.
  • An ester group is typically a group of the form - CO 2 R in which R is a C,-C 6 alkyl group as above.
  • a C r C 6 acyl group is typically a Ci, C 2 or C 3 acyl group.
  • antioxidant scavengers include compounds such as ascorbate, N-acetyl cysteine, cysteine, letosteine, procysteine, gamma glutamylcysteine ethyl ester, lipoic acid, 2- mercaptoethane sulphonate (mesna), ubiquinone, 6-hydroxynicotinic acid, vitamin E (also known as tocapherol), dithiolethiones (e.g.
  • anethole dithio (ADT or Sulfarlem from Solvay Pharma) or Oltipraz from Aventis), lansoproazole, MP-33 (Kalinina et al., 2002, Klin. Med, 80(5), p50-53), probucol, taurine, oxerutin, ergolines such as 6- hydroxynicotinic acid, stilbazulenyl nitrole, AGI-1067, lycopene, Trolox, mercaptopropionyllycine, tocopherols, carotenoids and polyphenols, particularly plant derived tocopherols, carotenoids and polyphenols, and derivatives which also exhibit antioxidant properties.
  • ADT anethole dithio
  • MP-33 Kalinina et al., 2002, Klin. Med, 80(5), p50-53
  • probucol taurine
  • oxerutin ergolines
  • ergolines such as 6- hydroxynicot
  • Prefe ⁇ ed examples include sulphur containing antioxidants (such as cysteine and its derivatives (e.g. N-acetyl cysteine and letosteine), lipoic acid and its derivatives (such as dihydrolipoic acid and mesna), ubiquinone, 6-hydroxynicotinic acid and Vitamin E.
  • Other prefe ⁇ ed antioxidants include Vitamin A.
  • Prefe ⁇ ed antioxidants or selections thereof may however exclude one or more of said antioxidants described above, e.g. may exclude ascorbic acid and/or lipoic acid.
  • Prefe ⁇ ed agents which act indirectly to increase antioxidant levels and/or oxidant defences include sulforaphane and derivatives thereof (such as 6-methyl sulfmylhexyl isothiocyanate (6-HITC), see Morimitsu et al, J. Biol. Chem., 2002, 277(5), p3456) which may also exhibit antioxidant properties, ethoxyquin and activators or mimics of enzymes of the glutathione oxidant defence system or phase 2 enzymes.
  • 6-HITC 6-methyl sulfmylhexyl isothiocyanate
  • agents that indirectly influence antioxidant levels and directly or indirectly influence oxidant defence levels include enzymes, their mimics and their activators. Particularly prefe ⁇ ed are those that catalyse the breakdown of ROS. Thus for example 4-phenyl butyrate (PBA) may be used which induces SOD or EUK 134 may be used (a synthetic SOD/catalase mimic).
  • PBA 4-phenyl butyrate
  • EUK 134 a synthetic SOD/catalase mimic
  • phase 2 enzyme inducers, particularly as described above also forms a prefe ⁇ ed aspect of the invention.
  • agents or selections thereof may however exclude one or more of said agents described above.
  • agents which are excluded are selected from the list consisting of estrogen or estrogen agonists which are estrogen receptor modulators (such as those flavones, flavonoids, estrogen and derivatives thereof and SERMS which have such properties).
  • agents also may exclude calcitonin and agonists which are calcitonin receptor modulators, PTH and agonists which are parathyroid-hormone receptor modulators such as PTHrP, Vitamin D and its derivatives, calcium and fluoride.
  • Said agents may also exclude SARMS (selective androgen-receptor modulators), osteoprotegerin, RANKL inhibitors, disintegrins, cysteine-protease inhibitors, H + -ATPase inhibitors, strontium salts, calcium receptor antagonists, low-density-lipoprotein-receptor-related protein 5 agonists, statins, leptin, growth hormone, insulin- like growth factor, fibroblast growth factor, transforming growth factor- ⁇ receptor, bone morphogenetic protein and bisphophonates (as for example described in Goltzman, 2002, supra).
  • SARMS selective androgen-receptor modulators
  • osteoprotegerin RANKL inhibitors
  • disintegrins cysteine-protease inhibitors
  • H + -ATPase inhibitors strontium salts
  • calcium receptor antagonists low-density-lipoprotein-receptor-related protein 5 agonists
  • statins leptin
  • growth hormone
  • Agents for use as hereinbefore described are conveniently provided in a pharmaceutical composition containing one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the agents in such compositions may be formulated as pharmaceutically acceptable salts.
  • the present invention also extends to pharmaceutical compositions comprising an agent as described hereinbefore, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.
  • “Pharmaceutically acceptable” as refe ⁇ ed to herein refers to ingredients that are compatible with other ingredients in the composition as well as physiologically acceptable to the recipient.
  • salts can be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Particularly prefe ⁇ ed salts include hydrochloric, hydrobromic, phosphoric, sulfuric, citric, maleic, citric and tartaric acid salts.
  • salts can be prepared from pharmaceutically acceptable non-toxic bases including inorganic or organic bases. Particularly prefe ⁇ ed salts are sodium, potassium and meglumine salts.
  • the present invention also extends to the use of such compositions and methods of prevention/treatment using such compositions, as described hereinbefore.
  • the agent may be administered orally, rectally, topically, Bacala, by inhalation or parenterally (e.g. intramuscularly, subcutaneously, intraperitoneally or intravenously) in the form of an injection or infusion. Patches, nasal sprays and chewing gum may also be used to administer the agent.
  • parenterally e.g. intramuscularly, subcutaneously, intraperitoneally or intravenously
  • Patches, nasal sprays and chewing gum may also be used to administer the agent.
  • the prefe ⁇ ed administration forms will be administered orally, rectally and by injection or infusion. The most prefe ⁇ ed administration form will be suitable for parenteral administration.
  • compositions according to the invention may be formulated in conventional manner using readily available ingredients.
  • the active ingredient may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • Biodegradable polymers such as polyesters, polyanhydrides, polylactic acid, or polyglycolic acid
  • the compositions may be stabilized by use of freeze-drying, undercooling or Permazyme.
  • Suitable excipients, ca ⁇ iers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpy ⁇ olidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.
  • compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • lubricating agents wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, adsorption enhancers, e.g. for nasal delivery (bile salts, lecithins, surfactants, fatty acids, chelators) and the like.
  • the active ingredient for administration may be appropriately modified for use in a pharmaceutical composition.
  • the active ingredient may be stabilized for example by the use of appropriate additives such as salts or non- electrolytes, acetate, SDS, EDTA, citrate or acetate buffers, mannitol, glycine, HSA or polysorbate.
  • Conjugates may be formulated to provide improved lipophilicity, increase cellular transport, increase solubility or allow targeting. These conjugates may be cleavable such that the conjugate behaves as a pro-drug. Stability may also be conferred by use of appropriate metal complexes, e.g. with Zn, Ca or Fe.
  • the pharmaceutical composition for oral use contains the active ingredient(s) and suitable physiologically acceptable agents to form tablets, capsules, solutions, suspensions or other well known formulations for oral administration.
  • suitable physiologically acceptable agents to form tablets, capsules, solutions, suspensions or other well known formulations for oral administration.
  • Such compositions can be prepared according to any method known for the manufacture of oral pharmaceutical compositions.
  • Such compositions can contain one or more agents as described hereinbefore and one or more agents selected from the group of preserving agents, inert diluents, viscosity increasing agents, colouring agents, sweetening agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, wetting agents, suspending agents, materials for preparation of delay formulations, oils and water.
  • compositions for other than oral use for example suppositories for rectal administration or solutions for injections or infusions can be prepared using well known methods and additives for such formulations. All formulations for injection and infusion should be sterile formulations. '
  • compositions may comprise from about 0.01% to about 99%o by weight of the formulation, preferably from about 0.1 to about 50%>, for example 10%.
  • the compositions are preferably formulated in a unit dosage form, e.g. with each dosage containing from about 0.0 lmg to about lg of the active ingredient, e.g. 0.05mg to 0.5g, for a human, e.g. 1-lOOmg.
  • the dose levels of the agent to be used are determined depending on a number of factors. The dose depends strongly on the choice of the agent, the clinical situation, the patient's age and weight and route of administration.
  • an agent as described hereinbefore can be combined with one or more other such agents to treat disorders as described herein, ie. bone loss disorders.
  • the agent can be combined with one or more other drugs with the same or different modes of action to treat the disorder.
  • examples of such combinations include the use of antioxidants in combination with one or more estrogen derivatives.
  • the invention extends to a composition comprising an agent as described hereinbefore together with one or more additional such agents and/or one or more additional active ingredients.
  • the invention further extends to use of such compositions and methods of using such compositions as described hereinbefore.
  • the invention further extends to a product comprising the components described above as a combined preparation for simultaneous, separate or sequential use in treating or preventing disorders as described hereinbefore.
  • a further aspect of the invention relates to the diagnosis of bone loss conditions.
  • the co ⁇ elation between a fall in available antioxidants, e.g. reduced and/or total glutathione levels, e.g. in tissue or blood cells, and bone loss, allows the levels of oxidant defences and/or antioxidants to be used as an indication of current or future bone loss.
  • the present invention further provides a method of diagnosing or monitoring a bone loss disorder in an individual, comprising determining the levels of oxidant defence and/or one or more antioxidants in a sample from said individual.
  • the levels of antioxidants in said sample are determined.
  • the level of total glutathione, reduced or oxidised glutathione, glutathione reductase or thioredoxin reductase is measured.
  • the sample is preferably a bone ma ⁇ ow or blood sample.
  • said sample comprises cells, preferably bone ma ⁇ ow cells such as osteoclasts, or blood cells, preferably monocytes or lymphocytes.
  • the levels determined in the individual may be compared to standard, known levels for healthy individuals and/or individuals suffering from the bone disorder under investigation.
  • diagnosis refers to the identification of a particular disorder in the patient under examination. Such diagnosis includes identification of a disorder prior to the appearance of other symptoms. Diagnosis also includes identification of patients at risk of developing such a disorder and/or providing a prognosis for such patients. “Monitoring” refers to establishing the severity of the disorder and/or the effects of treatment or the progression of the disorder.
  • mice Female 6-8-wk-old Wistar rats or MF1 mice or from the St George's colony or from Harlan Olac, Oxon, UK
  • mice Male 6-8-wk-old Wistar rats or MF1 mice or from the St George's colony or from Harlan Olac, Oxon, UK
  • ovariectomy or a sham operation followed by pair feeding.
  • pair feeding Three weeks later, a single dose of 17- ⁇ estradiol (10 ⁇ g/kg) or
  • 17- ⁇ estradiol (100 ⁇ g/kg) or vehicle was administered subcutaneously in corn oil. Animals were killed 24 hrs later. Success of ovariectomy was confirmed by absence of ovaries and atrophy of uteri (Chow et al, 1992, J. Clin. Invest., 89, p74-78). Femora were rapidly cleaned and bone marrow harvested into ice-cold heparinized water. Liver and spleen were weighed and homogenized in ice-cold water using a polytron homogenizer. The homogenates were divided into two equal parts. To one, 0.1 vol of 1%> Triton X100 was added; to the other an equal volume of 10% sulfosahcylic acid.
  • Triton extract was centrifuged at 10000 g for 10 min at 4°C and the supernatant was used for enzyme assays. Protein concentration was determined using Coomassie blue (Pierce, Tattenhall, Cheshire, UK) with bovine serum albumin as standard.
  • Glutathione was measured in the samples after deproteinisation with sulfosalycylic acid.
  • Total glutathione (GSH + GSSG) was measured using the GSH reductase-DTNB recycling procedure according to Tietze (Tietze et al., 1969, Analytic Biochemistry, 27, p502-522).
  • GSSG was assayed as above after derivatization of GSH in the sample with 2-vinylpyridine .(Baker et al. " , 1990, Analytic Biochemistry, 190, 360-365).
  • Glutathione reductase was assayed with a kit from Calbiochem (La Jolla, USA) according to the manufacturers instructions.
  • Thioredoxin and thioredoxin reductase were assayed by the NADPH-dependent reduction of DTNB at 412 nm in the insulin reducing assay (Holmgren et al, 1995, Methods in Enzymology, 252, pl99-208).
  • Glutathione reductase the enzyme responsible for regenerating glutathione from oxidised glutathione
  • thioredoxin reductase the enzyme responsible for regenerating thioredoxin
  • Figure IB ID
  • Thioredoxin levels also fell (Fig. 1C).
  • the levels of all these components of the thiol anti-oxidant system were rapidly normalized by a single dose of 17- ⁇ estradiol (10 ug/kg 5 the replacement dose of estradiol in rodents (Chow et al., 1992, supra))(Fig. 1A-D).
  • estrogen can be directly oxidant, and could thereby induce anti-oxidant defences.
  • glutathione, glutathione reductase and thioredoxin reductase were unchanged by ovariectomy in liver or spleen (data not shown), suggesting a receptor-mediated action in bone ma ⁇ ow.
  • the ability of ovariectomy to decrease, and a replacement dose of estrogen to normalize thiol anti-oxidants suggests that very low, physiological concentrations of estrogen are sufficient to maintain thiol anti-oxidants.
  • the equally-oxidant but receptor-inactive stereoisomer 17- ⁇ estradiol did not restore glutathione levels in rodent bone ma ⁇ ow (Fig. IE).
  • NAC and ascorbate increase tissue glutathione concentrations (Jain et al., 1992, PNAS USA, 89, p5093-5097). Their effect on bone loss was tested.
  • mice were subjected to ovariectomy or sham-ovariectomy. " Groups of six were administered NAC (100 mg/kg/day ip) or ascorbate (1 mmole/kg ip) or vehicle twice a day. For this, ascorbate was dissolved immediately before use in isosmolar ice-cold saline and adjusted to pH 6.8 with 2M NaOH. After 14 days animals were killed and bone prepared for analysis of static parameters of bone resorption and bone formation as described (Chow et al., 1992, supra).
  • FIGs. 2A and 3A show representative images of microscope sections of femora from mice 2 weeks after being subjected to sham ovariectomy (sham), ovariectomy (ovx), or ovariectomy with injections of NAC (Fig. 2A) or ascorbate (Fig. 3A).
  • NAC or ascorbate also normalized the number of osteoclasts per mm on the bone surface (Figs. 2C and 3C), the percentage of bone surface that was covered by osteoclasts (Figs. 2D and 3D) and the percentage of bone surface that showed a crenated, eroded surface characteristic of osteoclastic activity (Figs. 2E and 3E). NAC or ascorbate also reversed the ovariectomy-induced increase in osteoblast numbers (Figs. 2F and 3F) and the percentage of surface that was covered by osteoblasts (Figs. 2G and 3G). Ascorbate also increased total glutathione in mouse bone marrow (data not shown).
  • BSO is a specific inhibitor of glutathione synthesis. The effect of BSO on bone loss was therefore tested.
  • mice were administered BSO (2 mmol/kg ip) twice per day for 3 weeks. BSO was also included in the drinking water (20 mM). Calcein was injected one and six days before killing the animals. Bones were processed for static and dynamic analysis as described (Chow et al., 1992, supra).
  • FIG. 4A and B show representative sections of femora from a control mouse and a mouse injected with BSO twice a day for 3 weeks, showing loss of trabecula bone in BSO-treated mouse.
  • BSO caused substantial and significant loss of bone (Fig. 4C). Bone loss was accompanied by an increase in the number of osteoclasts per mm of bone surface, in the percentage of bone surface covered by osteoclasts, and in the percentage of bone surface that showed a crenellated, resorbed appearance (Fig. 4D- F).
  • BSO also significantly increased the number of osteoblasts covering bone surfaces, and the percentage of bone surface covered by osteoblasts (Fig. 4G, H). BSO caused a significant increase in the percentage of bone surface that was actively forming bone matrix (Fig. 41). However, the rate of deposition of the bone formed in actively-forming areas was not increased (Fig. 4J). There was an overall increase in the quantity of bone formed per unit time (Fig. 4K). Total glutathione fell significantly (p ⁇ 0.01, Student's t-test) in the bone ma ⁇ ow of BSO-treated mice from 14.5 ⁇ 1.1 to 6.8 ⁇ 0. lnmol mg protein. There was no significant change in body weight in either group of mice during the experimental period. Uterine weights did not differ significantly from control in BSO-treated mice.
  • Osteoclasts were generated from non-adherent murine bone ma ⁇ ow cells as previously described (Fuller et al., 2002, Endocrinology, 143, pi 108-1118). Briefly, after overnight incubation in M-CSF, non-adherent bone ma ⁇ ow cells (3xl0 5 per ml) were incubated in MEM/FBS with M-CSF (50 ng/ml) and RANKL (50 ng/ml) for 5 days.
  • BSO stimulated TRAP- positive multinucleate cell formation, while this was suppressed by NAC.
  • the ROS hydrogen peroxide also stimulated TRAP-positive multinucleate cell formation (Fig. 5C).
  • osteoclast cultures were washed and incubated in M-CSF plus BSO (100 uM) or NAC (30 mM) for 2 hrs before re- addition of RANKL. Cells were harvested by scraping 30 minuteslater.
  • Nuclear extracts were prepared as previously described (Partington et al., 1999, Nucleic acids Research, 27, pi 168-1175) except that the cysteine protease inhibitor trans- epoxysuccinyl-l-leucylamido(4-guanidino)-butane (Sigma, Poole, Dorset, UK), was included in the LS buffer (20 nM HEPES pH 7.9, 2 mM MgCl 2 ) at a final concentration of 3.0 nM and in addition microcystin-LR (Biogene-Alexis, Nottingham, UK) was included at a final concentration of 4.0 nM.
  • the cells were washed in situ twice with cold PBS, once with cold LS buffer and lyzed by the addition of LS buffer containing 0.1 %> Triton-X 100 with gentle scraping using a cell scraper.
  • the protein concentration in nuclear extracts was determined using the BCA protein assay reagent (Pierce) and EMSA was carried out using aliquots containing equal amounts of protein (4 ⁇ g/assay).
  • the NFKB p50 rabbit polyclonal antibody was supplied by Active Motif (Rixensart, Belgium). The following modifications were made to the previously published EMS A procedure (Partington et al., 1999, supra).
  • For the supershift assay the nuclear extracts were incubated for 30 min at 4°C with the antibody (2 ⁇ l/ assay) prior to the addition of the probe and poly(dldC).
  • the protein-DNA complexes were resolved on a 4% polyacrylamide gel in 0.5XTGE buffer for 2 hr at 4°C at 12.5 V/cm following the protocol recommended by Active Motif.
  • the NFKB oligonucleotide probe was supplied by Promega, 5 - AGTTGAGGGGACTTTCCCAGG.
  • the mutant NFKB oligonucleotide was supplied by Active Motif, 5*-GCCATGGGCCGATCCCCGAAGTCC. Binding of NFKB to the probe was increased by BSO and suppressed by NAC (Fig. 5D). The binding was confirmed to contain NFKB p50 by supershifting both bands with p50 antibody (a ⁇ ow). The binding was furthermore confirmed to be specific by competing binding of NFKB with an excess of unlabelled probe (S), but not by a mutant species (H).
  • osteoclasts were generated as above, washed, and medium replaced with phenol red-free MEM, charcoal-stripped serum and M-CSF, with 17- ⁇ estradiol (10 "9 M or vehicle).
  • RANKL 50 ng/ml
  • RNA was size-separated in a 1.2% agarose gel and blotted according to standard protocols. Blots were hybridized with 32 P-labelled probes for murine TNF- ⁇ and ⁇ -actin. Estrogen was observed to suppress expression of RNA for TNF- ⁇ in in vz ' tr ⁇ -osteoclasts. (Fig. 5e) *p ⁇ 0.05 vs control group. • . "
  • mice Groups of eight mice were subjected to ovariectomy or sham ovariectomy as described above (day 0). Two weeks later (day 14), a group of each treatment type was killed. One remaining group of ovarietomised mice was then administered ascorbate (Immole/kg/day ip) twice per day.
  • mice were sacrificed and bone volume measured as described above.
  • the bone volume of mice that had been administered ascorbate for 14 days was significantly greater (p ⁇ 0.05) than that of the mice killed 14 days after ovariectomy (figure 6).
  • the bone volume of ovariectomised mice after 28 days did not differ from the bone volume of ovariectomised mice after 14 days.

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Abstract

Selon l'invention, il est possible de traiter ou de prévenir les troubles associés à la perte osseuse par administration d'un agent qui accroît les taux de défenses contre les oxydants et/ou d'au moins un antioxydant chez un sujet. Ledit agent peut être un antioxydant. Cet agent peut agir directement pour accroître les taux d'antioxydant et/ou les défenses contre les oxydants. En variante, ledit agent peut agir indirectement.
PCT/GB2004/001695 2003-04-17 2004-04-19 Utilisation d'antioxydants pour traiter les troubles associes a la perte osseuse WO2004093995A2 (fr)

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WO2007130353A2 (fr) * 2006-05-01 2007-11-15 Johns Hopkins University Inducteurs de phase 2 et voies de signalisation associées protégeant le cartilage contre l'inflammation, l'apoptose et le stress
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