WO1998035674A1

WO1998035674A1 - Compositions for the inhibition of bone resorption

Info

Publication number: WO1998035674A1
Application number: PCT/GB1998/000443
Authority: WO
Inventors: Timothy Michael Skerry; Larry John Suva
Original assignee: Smith & Nephew Plc
Priority date: 1997-02-14
Filing date: 1998-02-12
Publication date: 1998-08-20
Also published as: AU6001798A; JP2001513757A; ZA981206B; EP0971712A1; CA2280722A1; GB9703109D0

Abstract

The disclosed invention is based on the observation that functional NMDA receptors are present on osteoclasts. Use of NMDA antagonists to inhibit bone resorption is disclosed, useful therefore in the treatment of bone disorders such as osteoporosis and Paget's disease. The NMDA antagonists used were Phencyclidine and Dizocilpine.

Description

COMPOSITIONS FOR THE INHIBITION OF BONE RESORPTION

The present invention relates to the field of bone biochemistry and is concerned in particular with the provision of medicaments for the treatment of diseases of the bone. The present invention is also concerned with novel usage for known materials and compositions and processes for the preparation of the same.

Vertebrate bone, as a tissue providing mechanical support for the body, undergoes constant remodelling through the formation and reso tion of bone mediated, it is widely thought, by the activities of osteoblasts and osteoclasts respectively. Bone remodelling comprises a complex and highly organised interaction between cells and the extracellular matrix (ECM). The remodelling process is, however, adaptive in response to requirements for growth or environmental stressing. In a normal healthy adult skeleton, the rate of bone formation approximates with the rate of bone resorption, a process known as coupling. Bone resorption or formation is not, though, a generalised feature of the skeleton but occurs in discrete sites which may be surrounded by areas of quiescent bone. Where resorption occurs excessively, several clinical problems can occur either at a specific locality or more extensively throughout the skeleton.

For example, osteoporosis is a disease that is characterised by abnormalities in the amount and architectural arrangement of bone tissue. Osteoporosis results from excessive resorption of bone and is a major clinical condition that can lead to fractures of bone following only minimal trauma. In addition to the distress to sufferers, the direct hospital costs of osteoporosis have been estimated, in the U.S. only, to approach $5.2 billion for women alone (Phillips et al (1988), Bone 9:271-279). The term Osteoporosis' in fact refers to a group of conditions that are associated with loss of bone tissue and an accompanying architectural abnormality that occurs in cancellous bone space. When the condition develops in post-menopausal women it is referred to as post menopausal osteoporosis. Fractures occur commonly in the hip, spine and distal radius and is considered by many countries to be a major public health problem (Lindasay R (1993), Clinical Rheumatology Osteoporosis; V.7, No.3). While diet and life-style appear to be factors in the pathogenesis of the disease, loss of ovarian function is an important determinant, at least in post menopausal osteoporosis.

Other osteoporotic-associated disease states include steroid induced osteoporosis, idiopathic juvenile osteoporosis, post- transplantation osteoporosis where bone resorption is a secondary indication of disorder.

In a disease of old age known as Paget's disease, there is excessive osteoclastic resorption of bone and reorganisation with loss of structure leading to deformities and liability to fracture.

Long term bed rest or disability for reasons that may not necessarily be directly related to diseases of the bone can lead to bone loss and danger of fracture on remobilisation or rehabilitation.

In cancer, osteoclastomas and other primary and secondary tumours often cause resorption and subsequent increased liability to fracture.

Tumour induced osteolysis may also lead to pathologically raised serum calcium levels which are believed to increase significantly morbidity in cancer patients.

Several approaches have been taken to limit bone resorption but none are entirely satisfactory with concern over undesirable side effects persisting. Oestrogen and other hormone replacements have a history of use, either alone or in combination with other therapeutics but suggestions of an increased risk of endometrial and breast cancer, as well as the continuation of menstrual bleeding, which is often unwelcome in the elderly female section of the population who form the majority of sufferers of osteoporosis, has provided a need for an alternative approach. The use of bisphosphonates is not ideal since the degree of side effects is regarded by some as unacceptably high and its use is not well tolerated by a significant proportion of the population.

In JP 63-295561 , 2-quinolone derivatives are disclosed which are described as having potent bone absorption-inhibiting activity.

As a result, there is an acute need to improve on the treatment of diseases or clinical conditions such as those outlined above which feature or are characterised by excessive or undesired bone resorption. It is therefore an object of the present invention to meet this need, or at least provide alternative approaches to those of the prior art.

N-methyl-D-Aspartate (NMDA) like receptor antagonists have a history of use in the field of medicine in treating disorders, primarily conditions such as neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycaemia and surgery as well as treating neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntingdon's disease, Down's syndrome, chronic pain, opiate tolerance and the like.

We have surprisingly found that NMDA like receptors, principally thought of as present in the central nervous system (CNS) where they are believed to be involved in processes such as learning and development are also present on the surface of at least osteoclasts, the cells mainly responsible for bone resorption and furthermore that the inhibitors of this receptor inhibit osteoclast activity.

Accordingly, in a first aspect of the present invention there is provided the use of a NMDA-receptor antagonist in the manufacture of a medicament for the treatment of diseases or clinical conditions featuring or characterised by undesired bone resorption.

In another aspect of the present invention, there is provided a method for the treatment of diseases or clinical conditions as hereinbefore described comprising the step of; administering a therapeutically effective amount of a NMDA-receptor antagonist.

In a further aspect of the present invention, there is provided a prophylactic method for the prevention of diseases or clinical conditions as hereinbefore described comprising the steps of repeated administration of a therapeutically effective amount of a NMDA-receptor antagonist.

In a further aspect, we provide the use of a NMDA-receptor antagonist in the manufacture of a medicament for the treatment of diseases of bone mass deficiency.

The term "NMDA-receptor antagonist" as used herein is meant a medicinal composition that inhibits the biological action triggered by the binding of glutamate or other natural ligand to a NMDA receptor or subtype thereof present on the surface of osteoclasts or other bone cell type. This may include, for example, reversible and irreversible competitive binding-site receptor inhibitors or other inhibitors (for example non-competitive inhibitor) that bind to a portion of the NMDA receptor and inhibits its activation for example glycine NMDA associated antagonists, glutamate site NMDA receptor antagonists and polyamine site antagonists. Other inhibitors include e.g. an NMDA receptor antibody or Fab fragment thereof or other protein moiety. Those skilled in the art will also recognise that this may include anti-sense mRNA capable of inhibiting translation of NMDA-receptor mRNA within osteoclasts.

The present invention is aimed at the treatment of undesired bone resorption which may often involve excessive resorption. The disease or clinical condition may feature bone resorption i.e. bone resorption is a secondary indication of the disorder. Alternatively, bone resorption may characterise, i.e. be the primary indication of the disorder.

Multi-nucleated osteoclasts are characterised by a high degree of expression of the enzyme Tartrate Resistant Acid Phosphatase (TRAP), an assay for which is disclosed in Barka et al: (1962) J Histochem Cytochem; 10,p741-753 and incorporated herein by reference.

A discussion of the classification of the glutamate family of receptors in given in Holimann et al (1994), Annu.Rev. Neurosci. 17;p31 to 108 which is incorporated herein by reference.

The receptor-antagonist of the present invention is preferably one which by virtue of its form (e.g. association with a carrier) and/or route of administration, does not cross to a significant extent the blood-brain barrier.

Clinical conditions and diseases of bone loss that may benefit from this invention include; osteoporosis, (including osteoporosis of disuse, Schϋller's disease, post menopausal osteoporosis, post- traumatic osteoporosis, senile osteoporosis), Paget's disease, undesired bone resorption featured in cancer, renal disease, fracture healing, rheumatoid arthritis and other arthritic conditions.

A receptor-antagonist previously used with respect to the CNS may be modified for use in the manufacture of medicament according to the present invention by for example modifying lipid solubility or increasing the charge on the receptor-antagonist such as known to those skilled in the art. Other possibilities include labile coupling of the receptor-antagonist to a 'bone-seeking' substance such as Tetracyclines, Bisphosphonates or antibody. Alternatively, NMDA-receptor antagonists previously thought of as having minimum therapeutic use because of their inability to penetrate the blood/brain barrier effectively to treat CNS disorders may be useful in the present invention. For example glycine NMDA associated antagonists such as 5,7 dichlorokynurenic acid and indole-2- carboxylic acid have been regarded as ineffectual as therapeutic agents for treating disorders associated with the NMDA receptor because of their inability to cross this barrier (McNarmara, D et al; Neurosci.Lett; 120;17-20 (1990) and Gray N.M et al, J.Med. Chem; 34 1283 to 1292 (1991). Accordingly, the NMDA receptor-antagonist for use in the present invention may be selected from a group consisting of, but not limited to;

Tartrates, maleates and phosphonopentanoic acids

Preferred tartrates include (7)-2-(4-benzyl piperidino-1)-p- hydroxyphenyl propanol tartrate (Cas No. 23210-58-4, Validex®, Hoechst Marion Roussel).

Preferred maleate include hydrogen maleates such as MK-801 hydrogen maleate (Merck, Sharpe and Dohme Inc)

Preferred phosphonopentanoic acids include D(-)-2-amino-5- phosphonopentanoic acid, (Research Biochemicals International RBI) and D,L-2-amino-5-phosphonovaleric acid (RBI)

Preferred glycine NMDA associated antagonists include various heterocyclic compounds and especially aromatic heterocyclic compounds. Glycine NMDA associated inhibitors of the invention can be selected from the group quinolones, quinoxalinediones, indole-2-carboxylates and 6-phenyl pyrazinones. The aromatic aryl ring preferably comprises a halo, e.g. chloro, substituted aryl ring.

Thus preferred quinolones are 7-chloro-2-quinolones, for example; 7-chloro-4-hydroxy-3-(3-phenoxyphenyl)-2(1 H)- quinolinone; 7-chloro-4-hydroxy-3-methoxycarbonyl-2-quinolone; or 7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl) pyridazino[4,5-b] quinoline-1 ,10(2H,5H)-dione monosodium salt.

Preferred quinoxalinediones are 7-chloro-quinoxaline-2,3- diiones for example; 7-chloro-6-methyl-5-nitro,14- dihydroquinoxaline; 6,7-dichloro-5-nitro-1 ,4-dihydroquinoxaline-2-3- dione;or (S)-9-chloro-5-[p-aminomethyl-o-(carboxy-methoxy)phenyl- carbamoylmethyl]-6,7-dihydro-1H,5H-pyridol[1 ,2,3-de]quinoxaline- 2,3-dione,e.g. the hydrochloride trihydrate.

Preferred indole-2-carboxylates for example, the Glaxo Welcome compound known as GV-150526A.

Preferred 6-phenyl pyrazinones are 6-(4-chlorophenyl) pyrazinones, for example 6-(4-chlorophenyl)imidazo[1 ,2- ajpyrazinone.

The receptor antagonists of the present invention are either known per se or may be manufactured according to the teaching of the art, e.g. the manufacture of MK801 is disclosed in US patent 4,399,141.

The medicament may be administered orally, subcutaneously, intra-muscularly, intravenously, either systematically but preferably locally, intraperitonealy or transdermally (e.g. skin patches) and may be in a composition form such as tablets, capsules, powders, granules, elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. Further composition forms includes pills and suppositories.

A therapeutically effective amount of the receptor-antagonist can be determined by routine observation and experimentation and will be dependent on factors such as the clinical condition or disease under treatment, age of the patient, severity and locality of the disease. For example, we have demonstrated that MK801 displays a dose-response relationship with concentrations of 10 μM showing minimal effect and concentrations of 100μM having a maximal effect in vitro.

Of course the therapeutically effective amount of the prophylactic prevention of a disease before clinical manifestation does not necessarily correspond to the effective amount for treatment of the clinical condition following manifestation. A therapeutically effective amount of the receptor-antagonist may be combined with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabiliser, flavour etc in a unit dosage form as called by accepted pharmaceutical practice. Pharmaceutical compositions of the present invention may comprise the antagonist together with an adjuvant.

Illustrative of the adjuvants which may be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacis, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose. A syrup or elixir may contain the receptor-antagonist, sucrose as a sweetening agent, methyl and propyl paralens as preservation, a dye and a flavouring.

Whilst the present invention is aimed primarily at humans, other animals e.g. mammals such as racehorses or dogs who suffer similar or related disorders of the bone may also benefit from the present invention.

The present invention will now be illustrated by way of example only.

1.Effect of NMDA antagonists on osteoclastogenesis In Vitro

Preparation of Osteoclast Osteoblast co-culture

An osteoclast/osteoblast co-culture was prepared as follows; calvaria from 12-20 new-born mice was dissected (2 to 3 days old Bb/CBA) and osteoblasts were isolated by digestion of the bones in collagenase followed by incubation in EDTA and a second collagenase digestion according to Takahashi U et al, (1988);

Endocrinology, 122,p.1373 to 1382 which is incorporated herein by reference. Cells were collected from each fraction and resuspended in alpha-MEM supplemented with 10% FCS and antibiotics (Gibco BRL, Paisley, UK) and kept on ice. The cells were then plated in 2 x 75 cm² flasks and after a minimum of one hour, non-adherent cells were removed and the medium replaced.

The long bones were dissected from 2 to 3 adult mice, and the marrow was flushed out with 2 to 4ml Hanks balanced salt solution supplemented with 10% FCS (Gibco/BRL, Paisley, UK) and using a 2SG needle. Pooled marrow sample were then passed through needles of increasing gauge (19G to 25G). Red cells were removed by centrifugation on a cushion of Ficoll-Paque (Pharmacist) at 600 x g for 25 mins. The marrow cells were harvested from the interface, washed once and resuspended in α-Minimum Essential Medium (MEM) supplemented with 10% FCS and antibiotics (Gibco). Adherent osteoblasts were resuspended by gentle trypsin treatment and co-cultures of 4 x 10⁴ osteoblasts together with 9 x 10⁵ bone marrow cells were prepared in wells of a 24 well plate, containing α-MEM with 10% FCS and 1,25(OH)₂D₃ (IO nM).

Cell number and viability was determined by MTT assay for mitochrondrial succinate dehydrogenase as disclosed in Journal of Immunological Methods; 168:253 to 256; 1994 and is incorporated herein by reference.

Test experiment

To test wells, test amount of known NMDA-receptor antagonist MK-801 (Merck) and Phencyclidine (PCP) were added for the times described to give the concentrations indicated.

To the control wells was added a volume of α-MEM equivalent to that in which the antagonist was prepared.

The formation of multinucleated osteoclasts was monitored after an eleven day period by the expression of high levels of tartrate resistant acid phosphatase (TRAP) and resorption in the mineralised dentine matrix on which they are cultured as outlined in Akatsu et_al (1992); V.7; 11 ; J. Bone and Min. Res which is incorporated herein by reference.

The above protocol was followed using Phencyclidine in place of MK-801.

Results

Co-cultures were maintained in the presence of MK-801 (100μM) for 11 days followed by TRAP staining. In control cultures, abundanr large, multinucleated TRAP-positive osteoclasts developed (Fig 1a and 1c) whereas MK-801 treated co-cultures, few TRAP-positive cells developed (Fig.1b) and those that did were predominantly mononuclear (Fig.ld). Both MK-801 and PCP showed dose-dependent effects on the area of TRAP stained cells over the 11 day culture period (Fig.2). Co-cultures experiments were performed where the cells were incubated on slices of dentine and exposed to NMDA antagonists MK-801 (Fig.3) and PCP (Fig.4) for varying durations. These experiments show that NMDA antagonists profoundly inhibit resorption.

Claims

1. Use of a NMDA-receptor antagonist in the manufacture of a medicament for the treatment of diseases or clinical conditions featuring or characterised by undesired bone resorption.

2. The medicament of claim 1 wherein the antagonist is a glutamate site antagonist

3. The medicament of claim 1 wherein the antagonist is a glycine site antagonist

4. The medicament of claim 1 wherein the antagonist is a glutamate ion channel antagonist.

5. The medicament according to any one of claims 1 to 3 wherein the antagonist is a reversible inhibitor.

6. The medicament according to claim 5 wherein the antagonist is a competitive inhibitor.

7. The medicament according to claim 5 wherein the antagonist is a non-competitive inhibitor.

8. The medicament according to any one of claims 1 to 4 wherein the antagonist is a irreversible inhibitor.

9. The medicament according to claim 1 wherein the antagonist is an antibody or Fab fragment thereof.

10. The medicament according to claim 1 wherein the antagonist is an anti-sense mRNA capable of inhibiting NMDA receptor mRNA translation within osteoclasts.

11. The medicament according to claim 1 wherein the antagonists is selected from a group consisting of; tartrates; maleates; phosphonopentanoic acids.

12. The medicament according to claim 10 wherein the maleate is a hydrogen maleate.

13. The medicament according to claim 3 wherein the glycine site antagonist is a heterocyclic compound.

14. The medicament according to claim 12 wherein the glycine site antagonist is selected from a group consisting of ; quinolones other than 2-quinolones; quinoxalinediones; indole-2-carboxylates; 6- phenyl pyrazinones.

15. The medicament according to claim 14 wherein the quinolone is a chloro-quinolone.

16. The medicament according to claim 14 wherein the quinoxalinedione is a chloro-quinoxalinedione.

17. The medicament according to claim 14 wherein pyrazinone is a chlorophenyl pyrazinone.

18. The medicament according to any preceding claim wherein the medicament comprises the antagonist together with a physiologically acceptable carrier therefor.

19. The medicament according to any preceding form in a form suitable for oral administration.

20. The medicament according to any preceding claim in a form suitable for injection.

21. A method for the treatment of a clinical condition featuring or characterised by undesired bone resorption in a mammalian patient comprising the step of administering a therapeutically effective amount of a NMDA-receptor antagonist.

22. A prophylactic method for the prevention of diseases or clinical conditions which feature undesired bone resorption comprising the steps of repeated administration of an effective amount of a NMDA- receptor antagonist.

23. A medicament for use in the treatment of diseases or clinical conditions featuring or characterised by undesired bone resorption comprising a NMDA-receptor antagonist.

24. A method according to any claim 21 or 22 wherein the disease is osteoporosis.