WO2004093995A2 - Use of antioxidants to treat bone loss disorders - Google Patents

Use of antioxidants to treat bone loss disorders Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
bone
glutathione
levels
agent
antioxidant
Prior art date
Application number
PCT/GB2004/001695
Other languages
French (fr)
Other versions
WO2004093995A3 (en
Inventor
Timothy John Chambers
Karen Fuller
Christopher John Jagger
Jenny Maree Lean
Original Assignee
St George's Enterprises Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St George's Enterprises Limited filed Critical St George's Enterprises Limited
Publication of WO2004093995A2 publication Critical patent/WO2004093995A2/en
Publication of WO2004093995A3 publication Critical patent/WO2004093995A3/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations

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.

Abstract

Bone loss disorders can be treated or prevented by administration of an agent which increases the levels of oxidant defences and/or at least one antioxidant in a subject. The agent can be an antioxidant and may act either directly to increase antioxidant levels and/or oxidant defences or indirectly.

Description

USE OF ANTIOXIDANTS TO TREAT BONE LOSS DISORDERS
Field of the Invention
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.
Background of the Invention
Bone maintains its strength via its ability to self renew, and turn over. This is achieved by constant resorption of bone in parallel with the formation of new bone.
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-κB 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).
In contrast to osteoclasts, 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.
The normally balanced processes of bone resorption and bone formation can become unbalanced in a number of different medical conditions, leading to the net loss of bone volume and/or density.
Normal bone loss usually starts between the ages of 20 and 35 years. An accelerated period of bone loss occurs over the 5 to 10 years following the menopause in women (early accelerated phase) before returning to a slower rate (slow phase). During the process of bone loss, the continued cycling of bone foπnation and resorption remains in place. The relative rates of bone resorption and formation are however affected by various factors, and mechanisms which have not yet been fully elucidated. Some of the factors involved have been identified, for example with increasing age the expression of osteoblast-modulating growth factors is affected. Hormonal effects also change with age, and therefore the loss of estrogens during the menopause is also a major causative effect for the imbalance between bone resorption and bone foπnation. Insufficient dietary intake of bone calcium causes increases in circulating parathyroid hormone (PTH). This may increase resorption of bone. Thus it can be seen that there are many factors which may affect the balance between bone formation and resorption.
Various diseases or disorders exhibit accelerated bone resorption and/or reduced bone formation leading to exacerbated bone loss.
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,Tιeparin 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.
Further types of generalised osteoporosis 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.
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.
The treatment and prevention of disorders associated with bone loss without causing significant adverse side effects remains a goal for practitioners in the art. A variety of approaches are cuπently in use for treating bone loss disorders. These may act to directly inhibit bone resorption, or may act anabolically to stimulate new bone formation.
It has been known for many years that postmenopausal osteoporosis is caused by estrogen deficiency. Administration of estrogen to postmenopausal women has been shown to improve calcium balance, and subsequently it was shown that the administration of estrogen prevented the accelerated bone loss that occurs following ovariectomy (reviewed in Riggs, supra).
It is not just postmenopausal osteoporosis that is dependent on estrogen deficiency. 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 β 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 foπnation.
Hormone replacement therapy, which involves the administration of oral forms of 17β 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 ,Δ8,9-dehydroestradiol, 17β,Δ8,9- dehydroestradiol and estra-5(10),7-dienes (such as estra-5(10),7-dien-3β-ol-17-ones, or its 3 sulphate ester, or estra-5(10),7 dien-3β-ol-17-one 3 glucuronides, estra- 5(10),7-dien-3α-ol-17-ones, or its 3 sulphate ester, or estra-5(10),7-dien-3α-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.
Further anti-resorptive agents of use in treating osteoporosis include SERMs (selective estrogen receptor modulators), see e.g. 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 (BMD) 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. The use of 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. However, 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. However the new bone produced is of poor quality and this leads to an increased number of fractures, and increased bone pain.
Full length PTH and fragments of this protein are anabolic agents that have also been suggested for use in the treatment of osteoporosis. The efficacy of this treatment is however dependent on the frequency of administration and the treatment has to be administered intermittently. Whilst PTH may be able to cause a substantial reversal of osteoporosis, a particular disadvantage of this treatment is that it requires administration by injection, and it induces osteosarcoma in rodents.
Generally, it is acknowledged in the art that whilst agents such as estrogen and bisphosphonate are able to prevent bone loss, only PTH has been shown to be effective in restoring lost bone or reversing bone loss. The drawbacks with using PTH are however described above.
Thus it can be seen that new and improved methods for treating or preventing disorders associated with bone loss, particularly osteoporosis, are required in view of the drawbacks of the current treatments available.
Summary of the Invention
Surprisingly it has now been found that a previously unrecognized family of agents have utility in the treatment or prevention of disorders associated with bone loss. It has been found that agents which result in increased levels of antioxidants and/or oxidant defences in the individual being treated have beneficial effects in treating or preventing bone loss in those individuals.
In the work leading up to this invention, the inventors have demonstrated, in an in vivo system, that the effect of ovariectomy and thus estrogen deficiency on bone loss could be mirrored by the administration of buthionine sulphoximide (BSO) which inhibits synthesis of the endogenous antioxidant, glutathione. The results are described hereinafter.
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 are routinely produced by cells. 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. Thus 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.
Many soluble proteins in cells contain sulphydryl groups, which need to be maintained in the reduced state. GSH assists this through reduction of oxidised glutaredoxin, which is then available to restore oxidised thiol groups on proteins to the reduced state. The reduced state of protein thiols is largely due to the activity of 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 foπn by thioredoxin reductase.
In addition, 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.
Moreover, some ROS, such as hydrogen peroxide, are highly diffusible and relatively stable. Hydrogen peroxide acts as a signalling molecule in cells. As mentioned above, the removal of hydrogen peroxide occurs through 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.
In the experiments conducted by the inventors, the administration of BSO to mice caused a decrease in total glutathione levels in bone maπow, loss of bone, accompanied by an increase in the number of osteoclasts per millimetre of bone surface, percentage of bone surface covered by osteoclasts and percentage of bone surface with a crenellated resorbed appearance. In parallel, there was also a significant increase in the number of osteoblasts covering bone surfaces and the percentage of bone surface covered by osteoblasts. A substantial increase in osteoclastic bone resorption parameters was seen, without a sufficient increase in bone formation to maintain overall bone volume.
The prevalence of endogenous antioxidants and the ability to generate such antioxidants was examined in mice subject to estrogen deficiency by ovariectomy which leads to bone loss. The direct effect of ovariectomy on the glutathione system was demonstrated by measuring reduced and oxidised glutathione, glutathione reductase activity, thioredoxin and thioredoxin reductase activity in bone maπow. The amount of glutathione and the activity of each of these enzymes was significantly reduced following ovariectomy. This was fully reversible by estrogen administration. Surprisingly however, the effects of ovariectomy on bone loss could also be fully reversed by the administration of the antioxidants, Vitamin C or N-acetyl cysteine.
The administration of Vitamin C at levels of 2 mmole/kg/day miπored 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.
Furthermore, when ovariectomised mice were administered ascorbate 14 days after ovariectomy, their bone volume was significantly greater than control mice who had been subjected to ovariectomy at the same time but without the subsequent administration of ascorbate. This demonstrates that administration of ascorbate can reverse bone loss in addition to preventing further bone loss.
These findings demonstrate for the first time that a link exists between the level of antioxidants which are present and the extent of bone formation/resorption. Whilst not wishing to be bound by theory, it is believed that the above results are indicative of the regulation of oxidant defences by estrogen and that estrogen deficiency may cause bone loss by lowering oxidant defences. This offers considerable scope for providing new and improved treatments for bone loss, e.g. osteoporosis, by administering agents that enhance oxidant defences, particularly the level of antioxidants.
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 confeπed 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.
Thus, no causal link has previously been shown between oxidant defences and bone loss, e.g. osteoporosis and in particular that increasing antioxidant levels and/or oxidant defences may have a beneficial effect on maintaining or improving bone integrity.
Thus 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:
(a) exposing bone cells or bone marrow cells to a candidate agent;
(b) assessing the ability of the candidate agent to increase the level of one or more of the components of the oxidant defence system; and
(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.
Brief Description of the Drawings
Figure 1 shows the levels of various components of the glutathione system in rat bone maπow following ovariectomy or sham treatment. Ovariectomised rats (ovx) were either treated with 17-β-estradiol (βE2), 17-α-estradiol (αE2) or left untreated, as indicated (A: a = GSH, b = GSSG);
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. The symbols are as shown in Figure 2 and dLS/BS = double-labelled bone surface/bone surface, MAR = mineral apposition rate, BFR/TBS = bone formation rate (μm3/day). *p<0.05 vs vehicle injected control. 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 (E2) on glutathione reductase (GR), thioreductase (TrXR) and glutathione, in osteoclasts. (c) shows the effect of BSO, NAC and H2O2 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 TNFα 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.
Detailed Description of the Invention
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.
As used herein "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. Thus for example, 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. For example, relative to a control, diseased, but untreated individual (or relative to the parameters at the start of 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. Relative to normal, undiseased individuals, 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. Thus 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.
In a particularly prefeπed embodiment, 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.
As used herein 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. Preferably 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. Most preferably 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.
As used herein, "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. Generally such antioxidants are free radical scavengers (scavenger antioxidants).
Such 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. Conveniently, 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.
Appropriate increases in 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:
1) antioxidant scavengers such as glutathione, alpha tocopherol, carotenoids, bilirubin and ascorbate;
2) enzymes and their cofactors which are involved in antioxidant processes (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) and other enzymes that act to remove ROS, with or without the help of other enzymes (e.g. superoxide dismutases, catalase, peroxidases); and
3) 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).
An increase in one or more of the above oxidant defences is envisaged in performing the method of the invention. 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%. Conveniently, 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. Appropriately such 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. Conveniently, 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.
Alternatively stated, 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.
Whilst not wishing to be bound by theory, one of the goals of the invention is to increase the tissue content of endogenous antioxidants (e.g. glutathione) in the reduced form. As mentioned previously, such endogenous antioxidants, particularly 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).
Therefore, 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. Thus, oxidant defences may be increased by the addition for example of high levels of antioxidants (scavenger antioxidants), preferably exogenous 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.
As mentioned above, 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.
As will be noted from the above comments, agents that increase the level of antioxidants will in effect increase the level of oxidant defences. 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.
As used herein, 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. Thus at its simplest, 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. Conveniently, antioxidant levels and/or oxidant defences are increased by activating the glutathione/ thioredoxin oxidant defence system (which is an important defence mechanism in bone maπow). Thus, preferably 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. Preferably 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. Preferably 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. Alternatively, 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, β-carotene, α-carotene, β-cryptoxanthin, zeaxanthin, lutein, echinenone, canthaxanthin and astaxanthin; carnosine and derivatives such as homocarnosine and anserine; carvedilol and metabolites, e.g. SB211475 and other β-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;
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.
Pharmacol., 438(3), p.189-196); diphenylphenylenediamine (DPPD); dithiolethiones (e.g. anethole dithio (ADT or Sulfarlem from Solvay Pharma) or
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
(synthetic SOD/catalase mimics, see Melor et al, 2001, J. Neurosci., 21(21), p.8348-
8353) or SOD mimics such as Mn2+- polyphosphate, -lactate, - succinate or -malate,
Desferal-Mn(IV) and related Mn complexes, copper complexes and TMPyP and other metalloporphyrins (e.g. AEOL 10113 and AEOL 10150, see Habeck, 2002,
DDT, 7(18), p.933); ergolines such as 6-hydroxynicotinic acid; ethoxyquin and related compounds including dihydroquino lines such as 6-ethoxy-
2,2-pentamethylen- 1 ,2-dihydroquinoline; flavanols, flavanones, flavanoids and chalcones and their denvatives, including wogonin, quercetin, resneratrol, silymarin (M2-80), carnosol, chrystin and carnosic acid and the flavonoids describerd in US Patents Nos. 5,849,786 and 6,528,042; flavones such as 4-bromoflavone, 4-chloroflavone, 4-trifluoromethylflavone, 3- bromoflavone and 3-chloroflavone; fullerenol (polyhydroxylated C(60)) derivatives such as hexasulfobutylated C60
(FC4S); 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
(e.g. NV-31, see Ahlemeyer et al., 2001, Brain Res., 890(2), p.338-42); gliclazide; 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.,
11(7), p.1189-1191);
GSH donors;
6-hydronicotinic acid;
4-hydroxytamoxifen;
6-hydroxy- 1 ,4-dimethyl-carbazole; indoles containing a triazole moiety; isoeugenol; isothiocyanates such as methylsulfinylalkyl isothiocyanates, such as sulforaphanes
(such as 4-methylsulfinylbutyl isothiocyanate or analogues such as 6- methylsulfinylhexylisothiocyanate); the aromatic isothiocyanates, including β- phenylethylisothiocyanate (PEITC) and benzyl isothiocyanate; and 7- methylsulfmylheptyl- and 8-methylsulfinyloctyl-isothiocyanates (Rose et al,
Carcinogenesis 21, 1983-1988, 2000); inhibitors of O2 " generation by phagocytes; inhibitors of phagocyte adhesion/respiratory burst; keto acids (such as pyruvate and α-ketoglutarate); ketoconazole; lactofeπin; lansoproazole; lazaroids, including U-74389G, U-83836E, U-101033, tirilazad mesylate, desmethytirilazad and related compounds and derivatives (see Karlsson et al., 2002,
Brain Res., 955(1-2), p.268-280); lipid-soluble chain-breaking antioxidants; lipoic acid;
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;
2 mercapto ethane sulphonate (mesna); mercaptoethylamine; mercaptopropionyllycine; mexiletine derivatives (e.g. MEX-NH, see Li et al., 2000, J. Pharmacol. Exp. Ther.,
295(2), p.563-571); MK-447;
MP-33 (Kalinina et al., 2002, Klin. Med, 80(5), p50-53);
N N-diphenyl-p-phenylene diamine; nitecapone; nitrile crambene (Νutr. Cancer 42, 233-240, 2002); non-calcineurin-binding analogues of FK506, especially GPI 1046 and V10367 (see
Tanaka et al., 2002, Neurosci. Lett., 321(1-2), p.45-48);
Oltipraz; omeprazole;
ONO-3144;
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; and gallic acid); plant pigments; probucol; promethazine; propofol; propyl gallate;
Pycnogenol (PYC); pyπolopyrimidines (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;
SOD/catalase/glutathione peroxidase mimics; some Ca2+ channel blockers; stobadine; sulforamate (4-methylsulfinyl-l-(S-methyldithiocarbamyl)-butane; Cancer Res. 57,
272-278, 1997); sumatriptan; synthetic metal-ion chelators (e.g. ICRF-187, hydroxypyridones); synthetic phenolic antioxidants, e.g. l-O-hexyl-2,3,5-trimethylhydroquinone; taurine; tempol; tetracyclines; thiols (e.g. mercaptopropionylglycine, N-acetylcysteine); tocopherols such as α-tόcopherol (or its water soluble form Trolox), 2-(alpha-D- glucopyranosyl)methyl-2,5,7,8-tetrachroman-6-ol, the vitamin E prodrug IRFI042
(see Attavilla et al., 2000, Cardiovasc. Res., 47(3), p.515-528) or analog
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.
Pharmacol, 406(2), p.281-292); triterpenes (e.g. celastrol); troglitazone; ubiquinone;
W-2721 and WR-1065 (amifostine), (see Grdina et al, 2002, Mil. Med., 167(2 suppl), p.51-53); xanthine oxidase inhibitors;
Zolimid; and molecules leading to induction of phase 2 proteins (which increase oxidant defences by activation of transcription factors that bind to the antioxidant response element of promoters of the relevant genes) for example as listed in Table 2 in Prestera et al,
1993, 1993, vol. 90, p2965 or in Dick & Kensler, 2002, Expert Rev. Anticancer
Ther., 2(5), p581-592, or certain chemoprotective agents.
Further suitable agents are the l,2-dithiole-3-thione derivatives disclosed in US Patents Nos. 5470871 and 5750560. These derivatives are compounds of formula (I) and (la):
Figure imgf000021_0001
Figure imgf000021_0002
in which
X is chosen from =S, =O, =N-OH, =N-R5, R5 being a CrC6 alkyl or an aryl group, =N-NH-CO-NH2 and =N-NH-CS-NH2, and
Z / =C \
Z'
Z and Z' being electron-attracting groups such as ester or cyano groups, A is chosen from a
\
C=N-OH group,
a group of foπnula
Figure imgf000021_0003
(where R3 is chosen from a C,-C6 alkyl group, a C,-C6 alkyl group substituted with one or more groups chosen from hydroxyl, amino, chloro and C,-C4 alkoxy groups, an aryl (C,-C6 alkyl) group, a (C C6 alkyl) carbonyl group and an aryl (C,-C6 alkyl) carbonyl group,
Figure imgf000021_0004
R4 being a C,-C6 alkyl group or an aryl group, and a CHOH group, Rj and R2 are chosen, independently of one another, from hydrogen, a halogen, a nitro group, a nitroso group, a thiocyano group, a CrC6 alkyl group, a C2- C6 alkenyl group, an aryl group, an aryl(CrC6 alkyl) group, an aryl(C2-C6 alkenyl) group, a carboxyl group, a (CrC6 alkyl)carbonyl group, an arylcarbonyl group, a (C C6 alkoxy)carbonyl group, a (CrC6 alkoxy)carbonyl(CrC6 alkyl) group, a C,-C6 alkoxy group, a trifluoromethyl group, an amino group, a di(CrC6 alkyl)amino(C,- C6 alkyl) group, an acylamino group of formula -NHCOCnH2n+1 with n from 0 to 6, a group -NH-CSCπH2n+1 with n from 0 to 6, a terpenyl group, a cyano group, a C2-C6 alkynyl group, a C2-C6 alkynyl group substituted with a CrC6 alkyl or an aryl group, ahydroxy(C,-C6 alkyl) group, a (CrC6 acyl)-oxy(CrC6 alkyl) group, a (CrC6 alkyl)thio group and an arylthio group, or alternatively Ry and R2 together form a mono- or polycyclic C2-C20 alkylene group optionally comprising one or more hetero atoms, optionally with the exception of the 2,2-dimethyltrimethylene group, or a C3-C12 cycloalkylene group,
R is chosen from a C C6 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
\ C=N— OH group,
a group of formula
Figure imgf000022_0001
(where R3 is chosen from a C]-C6 alkyl group, a C,-C6 alkyl group substituted with one more groups chosen from hydroxyl, amino, chloro and CrC6 alkoxy groups, an aryl(CrC6 alkyl) group, a (CrC6 alkyl)carbonyl group and an aryl (CrC6 alkyl) carbonyl group), a C^O group, a
\ C=N— R4 group,
R4 being a CrC6 alkyl group, and a CHOH group,
R, and R2 are chosen, independently of one another, from hydrogen, halogen, a CrC6 alkyl group, an aryl group, an aryl (CrC6 alkyl) group, a carboxyl group, an alkoxycarbonyl group, a CrC6 alkoxy group, a trifluoromethyl group, a di(C C6 alkyl) amino (CrC6 alkyl) group and an acylamino group of formula -NHCOCnH2n+I with n from 0 to 6, or alternatively Rj and R2 together from a C2-C12 alkylene group, optionally with the exception of the 2,2-dimethyltrimethylene group, or a C3-C12 cycloalkylene group,
R is chosen from a Cj-C6 alkyl group, and their pharmaceutically acceptable salts.
In the foregoing definitions, 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 C4 alkoxy group, a trifluoromethyl group, a nitro group and a hydroxyl group.
Other suitable agents are the five membered ring thiosulfinate esters and five-membered ring sulfenate esters of US Patent No. 6242478. The five membered ring of these esters has structure (II):
Figure imgf000023_0001
wherein, when X is O or S;
Rj is alkyl, preferably with 1-6 carbon atoms and more preferably methyl, phenyl, substituted phenyl, cyclohexenyl, or substituted cyclohexenyl;
R2 is methyl, phenyl or CO2R3 wherein R3 is alkyl .such as C,-C6 alkyl or phenyl; and, optionally, when X is O and R, is phenyl, R2 is not methyl.
Particularly suitable esters are compounds of formula (II) in which X is O, R{ is unsubstituted phenyl or methyl and R2 is CO2Me. In other prefeπed embodiments, when Rj is a substituted phenyl, the phenyl is substituted at the 4 position with a methoxy, t-butyl, alkyl ketone, sulfonamide, or trifluoromethyl group. Furthermore, when R is a substituted cyclohexenyl, the cyclohexenyl is preferably substituted in the 2 position with a halogen, an alkyl, or a phenyl. In certain prefeπed embodiments:
X is O, R[ is (4-Meo)phenyl, (4-t-Bu)phenyl or cyclohexenyl and R2 is methyl; and
X is O, R[ is phenyl, and R2 is alkyl, and preferably R2 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:
Figure imgf000024_0001
wherein,
R[ represents a hydrogen atom or a C,_3 alkyl group; and
R2 represents phenyl or furanyl group, or a group represented by the formula: -C(Ra)=C(Rb)(Rc), wherein Ra, Rb and Rc, being the same or different from each other, means a hydrogen atom or methyl or phenol group.
Among others, the compounds (III) wherein R[ is a hydrogen atom or methyl group and R2 is a group represented by the formula: -C(Ra)=C(Rb)(Ra) wherein Ra, Rb and Rc, being the same or different from each other, means a hydrogen atom or methyl group are prefeπed.
Additional suitable agents are 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. Prefeπed 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 (CH3CO(CH2)4NCS) (GHP 1105) and 6-isothiocyanato-2-hexanor(GHP 1106).
Further suitable antioxidants are disclosed in US Patent No. 6376498. These are the pyrazine derivatives of the formula (IV):
Figure imgf000024_0002
in which the radicals R1 to R9 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 (R5xR6)n wherein n>l, x represents one or more heteroatoms and R5 and R6 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,-C6 alkyl, alkenyl is typically C2-C6 alkenyl, alkinyl is typically C2-C6 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. Preferably, the radicals R2 to R9 and H and the radical R1 is a radical of the formula (V):
Figure imgf000025_0001
Suitable purine derivatives for use in the invention are the purines of US Patent No. 5801159. These purines have the following formula (VI):
Figure imgf000025_0002
wherein:
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 R1 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 Y1 to define a doubly bonded heteroatom;
Y and Y1 are hydrogen or may be taken together to form a double bond between the atoms to which they are bonded or Y1 may be taken together with R to form a double bond to the atom designated by R.
Various compounds coming within the formula are 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.
In the above definitions, a C,-C6 alkyl group is typically a CrC4 alkyl group or a C,-C3 alkyl group. Prefeπed alkyl groups are methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl and tert-butyl. A CrC6 alkoxy group is typically a C C4 alkoxy group. Prefeπed alkoxy groups are methoxy and ethoxy.
A C2-C6 alkenyl group is typically a C2 or C3 alkenyl group such as allyl. A C2-C6 alkynyl (or alkinyl) group is typically C2 or C3 alkynyl (or alkinyl) group. An aryl group is preferably phenyl. An ester group is typically a group of the form - CO2R in which R is a C,-C6 alkyl group as above. A CrC6 acyl group is typically a Ci, C2 or C3 acyl group.
The above described agents act through different processes to achieve an increase in oxidant defences and/or antioxidant levels.
Agents that directly influence antioxidant levels satisfy the test for antioxidants described hereinbefore. Prefeπed antioxidant agents are antioxidant scavengers and 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.
Prefeπed 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 prefeπed antioxidants include Vitamin A. Prefeπed 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.
Prefeπed 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.
As mentioned above, agents that indirectly influence antioxidant levels and directly or indirectly influence oxidant defence levels include enzymes, their mimics and their activators. Particularly prefeπed 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). The use of phase 2 enzyme inducers, particularly as described above also forms a prefeπed aspect of the invention.
Prefeπed agents or selections thereof may however exclude one or more of said agents described above. Preferably said 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). Preferably said 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-β receptor, bone morphogenetic protein and bisphophonates (as for example described in Goltzman, 2002, supra).
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.
Thus, 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 refeπed to herein refers to ingredients that are compatible with other ingredients in the composition as well as physiologically acceptable to the recipient.
If the agent is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Particularly prefeπed salts include hydrochloric, hydrobromic, phosphoric, sulfuric, citric, maleic, citric and tartaric acid salts.
If the agent is acidic, salts can be prepared from pharmaceutically acceptable non-toxic bases including inorganic or organic bases. Particularly prefeπed salts are sodium, potassium and meglumine salts. In further embodiments the present invention also extends to the use of such compositions and methods of prevention/treatment using such compositions, as described hereinbefore.
For the treatment and prevention of disorders as described herein, ie. bone loss disorders, especially osteoporosis, 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. The prefeπed administration forms will be administered orally, rectally and by injection or infusion. The most prefeπed administration form will be suitable for parenteral administration.
Pharmaceutical compositions according to the invention may be formulated in conventional manner using readily available ingredients. Thus, 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) may also be used for solid implants. The compositions may be stabilized by use of freeze-drying, undercooling or Permazyme.
Suitable excipients, caπiers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyπolidone, 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. The 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. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration of the patient by employing procedures well known in the art.
The active ingredient for administration may be appropriately modified for use in a pharmaceutical composition. For example, 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.
Thus for example, 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. 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.
Pharmaceutical 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.'
The active ingredient in such 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.
For treatment of disorders in accordance with the invention, 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.
According to a further aspect of the present invention, 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.
Alternatively, 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.
Thus 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 coπelation 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.
Thus 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. In one embodiment the levels of antioxidants in said sample are determined. Preferably the level of total glutathione, reduced or oxidised glutathione, glutathione reductase or thioredoxin reductase is measured. The sample is preferably a bone maπow or blood sample. Preferably said sample comprises cells, preferably bone maπow 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.
As refeπed to herein, "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.
The invention is further described in the following non-limiting Examples with reference to the following Figures in which: EXAMPLES
Example 1
Assessment of effect of ovariectomy and estrogen on oxidant defences
Groups of 6 rats or mice (female 6-8-wk-old Wistar rats or MF1 mice or from the St George's colony or from Harlan Olac, Oxon, UK) were subjected to ovariectomy or a sham operation, followed by pair feeding. Three weeks later, a single dose of 17-β estradiol (10 μg/kg) or
17-α estradiol (100 μg/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. The 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).
As can be seen from Figure 1, ovariectomy decreased glutathione in rat bone marrow, while administration of 17β estradiol restored glutathione in rat bone marrow (Fig. 1 A). This result implies a decrease in the thiol anti-oxidant capacity of bone marrow.
Glutathione reductase, the enzyme responsible for regenerating glutathione from oxidised glutathione, and thioredoxin reductase, the enzyme responsible for regenerating thioredoxin, were also substantially lower in bone maπow from ovariectomised rats (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-β estradiol (10 ug/kg5 the replacement dose of estradiol in rodents (Chow et al., 1992, supra))(Fig. 1A-D). An identical pattern of suppression of glutathione, glutathione reductase and thioredoxin reductase, and their normalization by estrogen, was also observed in the bone marrow of mice (data not shown). Thus, physiological levels of estrogen maintain thiol anti-oxidants in rodent bone maπow.
At high concentrations estrogen can be directly oxidant, and could thereby induce anti-oxidant defences. However, glutathione, glutathione reductase and thioredoxin reductase were unchanged by ovariectomy in liver or spleen (data not shown), suggesting a receptor-mediated action in bone maπow. Furthermore, 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. Lastly, the equally-oxidant but receptor-inactive stereoisomer 17-α estradiol did not restore glutathione levels in rodent bone maπow (Fig. IE). These results suggest that physiological levels of estrogen maintain thiol anti-oxidants in bone marrow through a receptor-mediated action.
Example 2
Effect of NAC and ascorbate on ovariectomy-induced bone loss
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).
NAC and ascorbate prevented bone loss in ovariectomised mice (Figs. 2,3). Both antioxidants normalized the ovariectomy-induced increase in osteoclast numbers, the extent of bone surface covered by osteoclasts, and the extent of eroded surface. Osteoblast indices were also restored to normal. Figures 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). The indices of bone resorption and bone formation show that while ovariectomy caused a reduction in bone volume (Figures 2B + 3B), this was prevented by NAC or ascorbate injections. 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).
Example 3
Effect of BSO on mouse bone
BSO is a specific inhibitor of glutathione synthesis. The effect of BSO on bone loss was therefore tested.
A group of 6 female 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).
BSO led to a fall in total bone maπow glutathione levels, and, like estrogen- deficiency, an increase in osteoclast numbers, eroded surface and bone loss (Fig. 4). 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 maπow 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. Example 4
Effects on in-vitro- generated osteoclasts
In order to identify the target cell on which these effects act, the effects of estrogen on osteoclasts were tested in vitro.
Osteoclasts were generated from non-adherent murine bone maπow cells as previously described (Fuller et al., 2002, Endocrinology, 143, pi 108-1118). Briefly, after overnight incubation in M-CSF, non-adherent bone maπow cells (3xl05 per ml) were incubated in MEM/FBS with M-CSF (50 ng/ml) and RANKL (50 ng/ml) for 5 days.
To test the effect of estrogen on thiol anti-oxidant mechanisms, osteoclasts were generated in 3x2 plates for 5 days. The cultures were washed and incubated for 18 hr in M-CSF, RANKL and 17-β estradiol (10"9M) or vehicle, in phenol red-free MEM and 10%> charcoal-stripped bovine serum. Cells were scraped and assayed for GSH, GSSG, glutathione reductase and thioredoxin reductase as described in Example 1 (n=9 for each). Estrogen stimulated glutathione reductase, thioredoxin reductase and glutathione in osteoclasts (Fig. 5A&B).
For assessment of the effect of agents on osteoclastic differentiation, non- adherent bone maπow cells were incubated with RANKL, M-CSF and BSO (10 uM), NAC (30 mM) or hydrogen peroxide (1 uM) (n=10). BSO stimulated TRAP- positive multinucleate cell formation, while this was suppressed by NAC. Like BSO, the ROS hydrogen peroxide also stimulated TRAP-positive multinucleate cell formation (Fig. 5C).
For assessment of NFKB activation, 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 MgCl2) 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 μg/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 μl/ 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 (aπow). 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).
For assessment of estradiol effects on TNF-α expression, osteoclasts were generated as above, washed, and medium replaced with phenol red-free MEM, charcoal-stripped serum and M-CSF, with 17-β estradiol (10"9M or vehicle). RANKL (50 ng/ml) was then added and cells were harvested for analysis of RNA after 3 hr of incubation. For this, 25-40 μg of total RNA was size-separated in a 1.2% agarose gel and blotted according to standard protocols. Blots were hybridized with 32P-labelled probes for murine TNF-α and β-actin. Estrogen was observed to suppress expression of RNA for TNF-α in in vz'trø-osteoclasts. (Fig. 5e) *p<0.05 vs control group. • . "
These results are compatible with the hypothesis that estrogen and thiol- modulating agents influence crucial ROS-sensitive signals in osteoclasts, the best- studied target for which is NFKB. BSO which reduces oxidant defences augmented the level of free NFKB whereas NAC suppressed that level consistent with reduced signalling through that molecule due to increase oxidant defences. TNF-α is a target gene for increased NFKB activity, and has also been implicated in estrogen- deficiency bone loss. The levels of TNF-α were also suppressed on administration of estrogen. The results above reveal a mechanism whereby estrogen deficiency augments ROS-sensitive signals such as NFKB, which in turn induce expression of cytokines such as TNFα, which are known to increase bone resorption. Example 5
Reversal of bone loss by ascorbate
The ability of ascorbate to reverse bone loss was also tested.
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.
At 28 days, the remaining 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.
This demonstrates that in addition to the ability of ascorbate to prevent bone loss, it may also reverse bone loss that has already occuπed.

Claims

1. Use of an agent which increases the levels of oxidant defences and/or at least one antioxidant in a human or non-human animal, in the manufacture of a medicament for treating or preventing a bone loss disorder in the human or non- human animal.
2. Use according to claim 1, wherein the disorder selected from rheumatoid arthritis, periodontitis, osteoporosis, Paget's disease, hypercalcaemia of malignancy, metastatic bone disease, multiple myeloma, osteogenesis imperfecta, osteomalacia, hyperparathyroidism and hypoarathyroidism.
3. Use according to claim 2, wherein the disorder is osteoporosis resulting from estrogen deficiency.
4. Use according to any one of the preceding claims, wherein the agent is an antioxidant.
5. Use according to any one of claims 1 to 3, wherein the agent acts directly to increase antioxidant levels and/or oxidant defences.
6. Use according to claim 5, wherein the agent is selected from ascorbate, N-acetyl cysteine, cysteine, letosteine, procysteine, gamma glutamylcysteine ethyl ester, lipoic acid, 2-mercaptoethane sulphonate, ubiquinone, 6-hydroxynicotinic acid, vitamin A, vitamin E, dithiolethiones, Oltipraz, lansoproazole, MP-33, probucol, taurine, oxerutin, ergolines, stilbazulenyl nitrole, AGI-1067, lycopene, Trolox, mercaptopropionyllycme, tocopherols, carotenoids and polyphenols.
7. Use according to any one of claims 1 to 3, wherein the agent acts indirectly to increase antioxidant levels and/or oxidant defences.
8. Use according to claim 7, wherein the agent is selected from sulforaphane and derivatives thereof which exhibit antioxidant properties, ethoxyquin and activators or mimics of enzymes of the glutathione oxidant defence system or phase 2 enzymes.
9. Use according to claim 8, wherein the sulforaphane derivative is 6- methylsulfinylhexyl isothiocyanate, 7-methylsulfmylheptyl isothiocyanate and 8- methylsulfinyloctyl isothiocyanate.
10. A method of screening for agents suitable for use in treating or preventing a bone loss disorder, which method comprises:
(a) exposing bone cells or bone marrow cells to a candidate agent;
(b) assessing the ability of the candidate agent to increase the level of one or more of the components of the oxidant defence system; and (c) determining whether or not the candidate agent may be suitable for said use.
11. A method according to claim 10, wherein osteoblasts or osteoclasts are employed in step (a).
12. A method according to claim 10 or 11, wherein the ability of the candidate agent to increase the level of one or more of glutathione, thioredoxin and their reductases is determined.
13. 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.
14. A method according to claim 13, wherein the level of glutathione, reduced or oxidised glutathione, glutathione reductase or thioredoxin reductase is measured.
15. A method according to claim 13 or 14, wherein the sample comprises bone maπow cells or blood cells.
16. A method of treating or preventing a bone loss disorder in a human or non-human animal, which method comprises the step of administering to the human or non-human animal an agent which increases the levels of oxidant defences and/or at least one antioxidant in the human or non-human animal.
17. An agent for treating or preventing a bone loss disorder in a human or non-human animal, comprising an agent which increases the levels of oxidant defences and/or at least one antioxidant in the human or non-human animal.
PCT/GB2004/001695 2003-04-17 2004-04-19 Use of antioxidants to treat bone loss disorders WO2004093995A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0308952.1 2003-04-17
GBGB0308952.1A GB0308952D0 (en) 2003-04-17 2003-04-17 Method

Publications (2)

Publication Number Publication Date
WO2004093995A2 true WO2004093995A2 (en) 2004-11-04
WO2004093995A3 WO2004093995A3 (en) 2005-04-21

Family

ID=9956987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2004/001695 WO2004093995A2 (en) 2003-04-17 2004-04-19 Use of antioxidants to treat bone loss disorders

Country Status (2)

Country Link
GB (1) GB0308952D0 (en)
WO (1) WO2004093995A2 (en)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006024096A1 (en) * 2004-08-31 2006-03-09 Chemgenex Pharmaceuticals Limited Method for modulating osteoclastogenesis
WO2007080630A1 (en) * 2006-01-10 2007-07-19 Masayoshi Yamaguchi AGENT FOR INCREASING BONE MINERAL CONTENT COMPRISING p-HYDROXYCINNAMIC ACID AS ACTIVE INGREDIENT
WO2007130353A2 (en) * 2006-05-01 2007-11-15 Johns Hopkins University Phase 2 inducers, ppar-alpha agonists and related signalling pathways protect cartilage against inflammation, apoptosis and stress
EP2046361A2 (en) * 2006-07-26 2009-04-15 The Regents of the University of California Osteogenic enhancer composition
EP2087902A1 (en) * 2008-02-05 2009-08-12 Bios Line S.p.a. Oral formulations designed to protect the airways, with special reference to inflammatory and neoplastic symptoms
WO2010001096A2 (en) * 2008-07-01 2010-01-07 Provexis Natural Products Limited Treatment
EP2148708A1 (en) * 2007-04-18 2010-02-03 McGill University Composition for enhancing bone formation
WO2010078322A1 (en) * 2008-12-30 2010-07-08 Hill's Pet Nutrition, Inc. Use of lipoic acid for treating or preventing degenerative joint conditions, osteoarthritis, cartilage damage, and related disorders in companion animals
US7956031B2 (en) 2005-05-31 2011-06-07 Naidu Lp Metallo-lactoferrin-coenzyme compositions for trigger and release of bioenergy
US8021659B2 (en) * 2006-04-28 2011-09-20 Naidu Lp Coenzyme Q10, lactoferrin and angiogenin compositions and uses thereof
CN102271529A (en) * 2008-12-30 2011-12-07 希尔氏宠物营养品公司 Companion animal compositions including lipoic acid and methods of use thereof
KR101169850B1 (en) * 2008-12-30 2012-07-30 서울대학교산학협력단 A pharmaceutical composition for preventing and treating bone metabolic disease or metastatic cancer by bone metastasis comprising Trolox
CN103826632A (en) * 2011-08-26 2014-05-28 国立大学法人名古屋大学 Osteogenesis promoter and use thereof
US20150031701A1 (en) * 2007-03-12 2015-01-29 St Ip Holding Ag Compositions and methods for preventing and treating mucositis and weight loss
WO2016187394A1 (en) 2015-05-19 2016-11-24 Van Wijnen Andre J Methods and materials for promoting bone formation
WO2017120420A1 (en) * 2016-01-06 2017-07-13 The Trustees Of Columbia University In The City Of New York The use of guaiacol for the prevention and treatment of glycogen storage disease
CN112089719A (en) * 2020-09-07 2020-12-18 广州中医药大学第一附属医院 Application of troxerutin in preparation of bone formation related medicines
CN112469279A (en) * 2018-07-09 2021-03-09 洛桑大学 Fungicides for the prevention and control of fungal pathogens
CN112972475A (en) * 2021-03-24 2021-06-18 南京医科大学友谊整形外科医院有限责任公司 Application of oltipraz in preparation of product for preventing and treating osteoporosis

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4590067A (en) * 1984-10-18 1986-05-20 Peritain, Ltd. Treatment for periodontal disease
JPH0687750A (en) * 1992-09-08 1994-03-29 Taiyo Yakuhin Kogyo Kk Medicine composition
JPH0840888A (en) * 1994-07-28 1996-02-13 Ajinomoto Co Inc Antitumor agent against myeloma-based tumor
JPH0881380A (en) * 1994-09-09 1996-03-26 Suntory Ltd Anti-periodontitis agent and anti-periodontitis food containing high-molecular weight polyphenol as active component
JPH11130644A (en) * 1997-10-29 1999-05-18 Lion Corp Composition for oral cavity
CA2012208C (en) * 1989-03-27 2001-05-22 Julius Ozick Method of treating periodontitis
US20020048798A1 (en) * 2000-03-15 2002-04-25 Avery Mitchell Allen Novel antioxidants
EP1214893A1 (en) * 2000-12-16 2002-06-19 Aventis Pharma Deutschland GmbH Health promoting compositions
EP1431385A1 (en) * 2002-12-18 2004-06-23 Döhler GmbH Xanthohumol containing beverage

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4590067A (en) * 1984-10-18 1986-05-20 Peritain, Ltd. Treatment for periodontal disease
CA2012208C (en) * 1989-03-27 2001-05-22 Julius Ozick Method of treating periodontitis
JPH0687750A (en) * 1992-09-08 1994-03-29 Taiyo Yakuhin Kogyo Kk Medicine composition
JPH0840888A (en) * 1994-07-28 1996-02-13 Ajinomoto Co Inc Antitumor agent against myeloma-based tumor
JPH0881380A (en) * 1994-09-09 1996-03-26 Suntory Ltd Anti-periodontitis agent and anti-periodontitis food containing high-molecular weight polyphenol as active component
JPH11130644A (en) * 1997-10-29 1999-05-18 Lion Corp Composition for oral cavity
US20020048798A1 (en) * 2000-03-15 2002-04-25 Avery Mitchell Allen Novel antioxidants
EP1214893A1 (en) * 2000-12-16 2002-06-19 Aventis Pharma Deutschland GmbH Health promoting compositions
EP1431385A1 (en) * 2002-12-18 2004-06-23 Döhler GmbH Xanthohumol containing beverage

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBASE [Online] ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL; 1994, HANIOKA T ET AL: "Effect of topical application of Coenzyme Q10 on adult periodontitis" XP002291089 Database accession no. EMB-1995041314 & MOLECULAR ASPECTS OF MEDICINE 1994 UNITED KINGDOM, vol. 15, no. SUPPL., 1994, pages S241-S248, ISSN: 0098-2997 *
HALL T J ET AL: "THE ROLE OF REACTIVE OXYGEN INTERMEDIATES IN OSTEOCLASTIC BONE RESORPTION" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 207, no. 1, 6 February 1995 (1995-02-06), pages 280-287, XP002068360 ISSN: 0006-291X *
NAKAMUTA HIROMICHI ET AL: "A novel bone-protective effect of taurine on low Ca diet-induced osteoporosis in rats" JAPANESE JOURNAL OF PHARMACOLOGY, vol. 88, no. Supplement 1, 2002, page 108P, XP009034443 & 75TH ANNUAL MEETING OF THE JAPANESE PHARMACOLOGICAL SOCIETY; KUMAMOTO, JAPAN; MARCH 13-15, 2002 ISSN: 0021-5198 *
PATENT ABSTRACTS OF JAPAN vol. 0183, no. 47 (C-1219), 30 June 1994 (1994-06-30) & JP 6 087750 A (TAIYO YAKUHIN KOGYO KK), 29 March 1994 (1994-03-29) *
PATENT ABSTRACTS OF JAPAN vol. 1996, no. 06, 28 June 1996 (1996-06-28) & JP 8 040888 A (AJINOMOTO CO INC), 13 February 1996 (1996-02-13) *
PATENT ABSTRACTS OF JAPAN vol. 1996, no. 07, 31 July 1996 (1996-07-31) & JP 8 081380 A (SUNTORY LTD), 26 March 1996 (1996-03-26) *
PATENT ABSTRACTS OF JAPAN vol. 1999, no. 10, 31 August 1999 (1999-08-31) & JP 11 130644 A (LION CORP), 18 May 1999 (1999-05-18) *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006024096A1 (en) * 2004-08-31 2006-03-09 Chemgenex Pharmaceuticals Limited Method for modulating osteoclastogenesis
US9283253B2 (en) 2005-05-31 2016-03-15 Naidu Lp Metallo-lactoferrin-coenzyme compositions for detoxification support
US7956031B2 (en) 2005-05-31 2011-06-07 Naidu Lp Metallo-lactoferrin-coenzyme compositions for trigger and release of bioenergy
US8476223B2 (en) 2005-05-31 2013-07-02 Naidu Lp Metallo-lactoferrin-coenzyme compositions to improve sleep patterns
US9180148B2 (en) 2005-05-31 2015-11-10 Naidu Lp Metallo-lactoferrin-coenzyme compositions and methods for weight management
WO2007080630A1 (en) * 2006-01-10 2007-07-19 Masayoshi Yamaguchi AGENT FOR INCREASING BONE MINERAL CONTENT COMPRISING p-HYDROXYCINNAMIC ACID AS ACTIVE INGREDIENT
WO2007080659A1 (en) * 2006-01-10 2007-07-19 Masayoshi Yamaguchi AGENT FOR INCREASING BONE MINERAL CONTENT COMPRISING p-HYDROXYCINNAMIC ACID AS ACTIVE INGREDIENT
US8021659B2 (en) * 2006-04-28 2011-09-20 Naidu Lp Coenzyme Q10, lactoferrin and angiogenin compositions and uses thereof
US8968726B2 (en) 2006-04-28 2015-03-03 Naidu Lp Lactoferrin and angiogenin compositions and uses thereof
WO2007130353A2 (en) * 2006-05-01 2007-11-15 Johns Hopkins University Phase 2 inducers, ppar-alpha agonists and related signalling pathways protect cartilage against inflammation, apoptosis and stress
WO2007130353A3 (en) * 2006-05-01 2008-05-29 Univ Johns Hopkins Phase 2 inducers, ppar-alpha agonists and related signalling pathways protect cartilage against inflammation, apoptosis and stress
JP2009544411A (en) * 2006-07-26 2009-12-17 ザ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・カリフォルニア Bone formation enhancing composition
EP2046361A2 (en) * 2006-07-26 2009-04-15 The Regents of the University of California Osteogenic enhancer composition
EP2046361A4 (en) * 2006-07-26 2011-06-29 Univ California Osteogenic enhancer composition
US20150031701A1 (en) * 2007-03-12 2015-01-29 St Ip Holding Ag Compositions and methods for preventing and treating mucositis and weight loss
EP2148708A1 (en) * 2007-04-18 2010-02-03 McGill University Composition for enhancing bone formation
EP2148708A4 (en) * 2007-04-18 2012-12-05 Univ Mcgill Composition for enhancing bone formation
EP2087902A1 (en) * 2008-02-05 2009-08-12 Bios Line S.p.a. Oral formulations designed to protect the airways, with special reference to inflammatory and neoplastic symptoms
WO2010001096A3 (en) * 2008-07-01 2010-04-08 Provexis Natural Products Limited Treatment
WO2010001096A2 (en) * 2008-07-01 2010-01-07 Provexis Natural Products Limited Treatment
US8952052B2 (en) 2008-12-30 2015-02-10 Hill's Pet Nutrition, Inc. Use of lipoic acid for treating or preventing degenerative joint conditions, osteoarthritis, cartilage damage, and related disorders in companion animals
KR101169850B1 (en) * 2008-12-30 2012-07-30 서울대학교산학협력단 A pharmaceutical composition for preventing and treating bone metabolic disease or metastatic cancer by bone metastasis comprising Trolox
EP2653161A1 (en) * 2008-12-30 2013-10-23 Hill's Pet Nutrition, Inc. Use of lipoic acid for maintaining or increasing bone mineral density in companion animals
WO2010078322A1 (en) * 2008-12-30 2010-07-08 Hill's Pet Nutrition, Inc. Use of lipoic acid for treating or preventing degenerative joint conditions, osteoarthritis, cartilage damage, and related disorders in companion animals
US9271957B2 (en) 2008-12-30 2016-03-01 Colgate-Palmolive Company Methods of treating or preventing degenerative joint conditions, osteoarthritis, cartilage damage, and related disorders in companion animals
CN102271529A (en) * 2008-12-30 2011-12-07 希尔氏宠物营养品公司 Companion animal compositions including lipoic acid and methods of use thereof
CN103826632B (en) * 2011-08-26 2017-04-12 国立大学法人名古屋大学 Osteogenesis promoter and use thereof
CN103826632A (en) * 2011-08-26 2014-05-28 国立大学法人名古屋大学 Osteogenesis promoter and use thereof
US20140343104A1 (en) * 2011-08-26 2014-11-20 National University Corporation Nagoya University Osteogenesis promoter and use thereof
WO2016187394A1 (en) 2015-05-19 2016-11-24 Van Wijnen Andre J Methods and materials for promoting bone formation
EP3310372A4 (en) * 2015-05-19 2019-03-06 Mayo Foundation For Medical Education And Research Methods and materials for promoting bone formation
WO2017120420A1 (en) * 2016-01-06 2017-07-13 The Trustees Of Columbia University In The City Of New York The use of guaiacol for the prevention and treatment of glycogen storage disease
US10933031B2 (en) 2016-01-06 2021-03-02 The Trustees Of Columbia University In The City Of New York Use of guaiacol for the prevention and treatment of glycogen storage disease
CN112469279A (en) * 2018-07-09 2021-03-09 洛桑大学 Fungicides for the prevention and control of fungal pathogens
CN112089719A (en) * 2020-09-07 2020-12-18 广州中医药大学第一附属医院 Application of troxerutin in preparation of bone formation related medicines
CN112972475A (en) * 2021-03-24 2021-06-18 南京医科大学友谊整形外科医院有限责任公司 Application of oltipraz in preparation of product for preventing and treating osteoporosis

Also Published As

Publication number Publication date
GB0308952D0 (en) 2003-05-28
WO2004093995A3 (en) 2005-04-21

Similar Documents

Publication Publication Date Title
WO2004093995A2 (en) Use of antioxidants to treat bone loss disorders
US6416737B1 (en) Increasing bone strength with selected bisphosphonates
Lal et al. Role of oxidative stress in advanced glycation end product-induced mesangial cell activation
McCormick et al. Effects of ovariectomy and estrogen on skeletal muscle function in growing rats
Palmer et al. Dietary antioxidant supplementation reduces lipid peroxidation but impairs vascular function in small mesenteric arteries of the streptozotocin-diabetic rat
Yamasoba et al. Role of glutathione in protection against noise-induced hearing loss
Bhatti et al. Mechanisms of antioxidant and pro-oxidant effects of α-lipoic acid in the diabetic and nondiabetic kidney
EP0801564B1 (en) Use of nebivolol as an anti-atherogenic
US6133322A (en) Quinone derivatives for treating or preventing diseases associated with iron overload
Poncin et al. Oxidative stress: a required condition for thyroid cell proliferation
Picardo et al. Antioxidant status in the blood of patients with active vitiligo
US20190060336A1 (en) Resetting biological pathways for defending against and repairing deterioration from human aging
JP4256679B2 (en) How to treat restenosis
Collinsova et al. Inhibition of betaine-homocysteine S-methyltransferase causes hyperhomocysteinemia in mice
Sakr et al. Effect of selenium on carbimazole-induced histopathological and histochemical alterations in prostate of albino rats
US20020147155A1 (en) Prevention and treatment of endometriosis with aryl hydrocarbon receptor binding ligands
Poršová-Dutoit et al. Do DHEA/DHEAS play a protective role in coronary heart disease
ES2431085T3 (en) Use of glutamate, glutamate derivatives or metabolites, glutamate analogues or mixtures thereof for the manufacture of a composition for the treatment of osteoporosis
Thokala et al. Loss of ErbB2-PI3K/Akt signaling prevents zinc pyrithione-induced cardioprotection during ischemia/reperfusion
Toba et al. The direct antioxidative and anti-inflammatory effects of peroxisome proliferator-activated receptors ligands are associated with the inhibition of angiotensin converting enzyme expression in streptozotocin-induced diabetic rat aorta
Liu et al. Oral nicotine administration decreases tumor necrosis factor-alpha expression in fat tissues in obese rats
JPS61500496A (en) Use of cysteine derivatives or their salts to increase insulin secretion from the islets of Langerhans in the pancreas
Guven et al. Effect of 3-amino benzamide, a poly (adenosine diphosphate-ribose) polymerase inhibitor, in experimental caustic esophageal burn
FR3125409A1 (en) Food supplement to fight female and male infertility
KR100304312B1 (en) Zinc Supplemented Prostate Extract

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase