WO2000067736A2

WO2000067736A2 - Therapeutic use of an inhibitor or an antagonist of an abc protein in bone

Info

Publication number: WO2000067736A2
Application number: PCT/GB2000/001736
Authority: WO
Inventors: James Anthony Gallagher; Wayne Barry Bowler; Simon Christopher Wagstaff
Original assignee: The University Of Liverpool
Priority date: 1999-05-07
Filing date: 2000-05-05
Publication date: 2000-11-16
Also published as: GB9910693D0; JP2003501350A; AU4592000A; EP1223943A2; WO2000067736A3

Abstract

Use of a compound which will act as an inhibitor or antagonist of the expression or function of an ABC protein, and a method of screening for a compound which will act as an inhibitor or antagonist of the expression or function of an ABC protein.

Description

DESCRIPTION

A COMPOUND FOR USE IN MEDICINE

The present invention relates to the use of a compound and composition

which will act as an inhibitor or antagonist of the expression or function of an ABC

protein in bone, more particularly an osteoclast associated ABC protein, for use in

medicine, and more particularly to the use of a compound and composition which

will act as an inhibitor or antagonist of the expression or function of an ABC protein,

more particularly an osteoclast associated ABC protein for use in the manufacture of

a medicament for use in the treatment of a disease where full or partial inhibition of

bone resorption will result in an improvement in the disease. Such diseases include,

but are not limited to, osteopenia, such as osteoporosis, Paget's disease, bone

metastases, myeloma, periodontal disease and humoral hypercalcaemia of

malignancy.

The invention also relates to a method of screening for a compound which

will act as an inhibitor or antagonist of the expression or function of an ABC protein

in bone, more particularly an osteoclast associated ABC protein, comprising

determining whether the presence of said compound leads to full or partial inhibition

of bone resorption.

ABC proteins (ATP binding cassette proteins), also called traffic ATP ases,

are a super family of transmembrane proteins involved in the movement of substrates

across cell membranes. ABC proteins are abundant in prokaryotes where they represent almost 5% of the total genome and show specificity for a diverse range of

substrates ranging from peptides and amino acids to ions and sugars. ABC proteins

are characterised by the presence of 2 peptide motifs, Walker A and Walker B motifs.

These motifs are common to many nucleotide binding proteins. However, ABC

proteins are distinguished from these other proteins by the presence of a third C-

signature motif, separating Walker A and B motifs with conserved spacing.

The importance of ABC proteins in mammalian systems is now being

recognised. Several members of the ABC family have been shown to be important

in human disease, their dysfunction results in a variety of disease states including

cystic fibrosis, multi-drug resistance of tumour cells, non-insulin dependent diabetes

and adrenoleukodystrophy. These ABC proteins are known to be involved in the

translocation of ion and hydrophobic drugs across the plasma membrane. Other

human ABC proteins have also been shown to be involved in peptide translocation

(PAB) and phospholipid transfer across the canalicular membrane, as is the case with

the MRP sub family. In addition, human ABC - 1 has recently been implicated as a

regulator of phospholipid equilibrium and non-classical (signal independent)

secretion of IL1- β in macrophages.

The applicant has discovered ABC transporter proteins in bone and osteoclast

rich tissue and identified several novel members of the ABC protein family from

osteoclastoma cDNA libraries and human bones cDNA libraries by immuno-

screening and hybridrisation screening.

The applicant's discovery has indicated that compounds which will either inhibit or promote expression or function of an ABC protein, more particularly an

osteoclast associated ABC protein, may be useful in treating conditions arising out of osteoclastic function.

This role of ABC proteins, more particularly an osteoclast specific protein has

not previously been identified.

However, in view of the fact that P-glycoprotein has recently been found to

be present in osteoblasts (Calcified Tissue International, 1996 Vol 58 No. 3 PI 86-

191) one can postulate that members of the ABC family of proteins may be involved

in bone formation.

More generally, ABC cassette proteins have been implicated in many cellular

functions. As such an osteoclast associated family of ABC transporters may regulate

many processes within these cells.

The ability of members of the ABC super family to regulate volume-activated

channels via ATP release has been documented in other cell types.

Osteoclasts are terminally differentiated cells and are hence programmed to

die by apoptosis. The ABCl member of the ABC superfamily has been implicated

in the recognition of apoptotic cells by macrophages, a process thought to involve

transmembrane flux of phosphatidylserine.

Inhibition or promotion of some of these putative functions of ABC proteins

expressed in bone and osteoclasts is indicated as having complex effects on, for

example, bone resorption ranging from inhibition, through no effect, to stimulation.

Inhibition of osteoclast apoptosis, would lead to a subsequent elevation in the functional osteoclast pool and enhanced resorption. Similarly, sulphonylurea

sensitivity is conferred on K/ATP channels through the presence of ABC

transporters, inhibition of which blocks potassium ion efflux and consequent calcium

ion influx thereby promoting insulin secretion. It is therefore indicated that blocking

osteoclast associated ABC transporters associated with ion channels will enhance

release of factors that may stimulate resorption, including regulatory factors, protons

and proteases including Cathepsin K. Conversely, the processes of osteoclast fusion

from mononuclear precursors, and those of cellular adhesion, if blocked will lead to

decreased resorption. As suggested earlier, transmembrane phospholipid trafficking

by the ABC 1 transporter provides a mechanism for osteoclast fusion, whilst cellular

adhesion may be promoted by annexin-mediated binding between phospholipids and

extracellular matrix.

The applicant has gone on to convincingly demonstrate that inhibition of

ABC proteins using Glibenclamide, a known inhibitor of known ABC proteins, in

osteoclast containing populations inhibits resorption. These results demonstrate that

inhibitors or antagonists of ABC proteins will be useful therapeutic agents in the

therapy of diseases where inhibition of resorption is desirable. These include

osteopenia, which includes osteoporosis, Paget's disease, bone metastases, myeloma

periodontal disease and humoral hypercalcaemia of malignancy.

Additionally, the applicant has surprisingly found that parathyroid hormone

(PTH), a known stimulant of bone resorption, when present with compounds of the

present invention, enhances the inhibitory effect of the compound. According to a first aspect of the present invention there is provided a

compound which will act as an inhibitor or antagonist of an ABC protein in bone for

use in the manufacture of a medicament for use in the treatment of a disease where

full or partial inhibition of bone resorption will result in an improvement in the

disease.

Examples of existing compounds which have similar action to the sulphonyl

urea, Glibenclamide, include:

TOLBUT AMIDE,

CHLOROPROPAMIDE,

TOLOZAMIDE,

GLIPIZIDE,

GLIQUIDONE, and

GLICLAZIDE.

The compounds may be administered orally, intravenously, subcutaneously

or by any other traditional route. For oral application, the compounds of the present

invention can be administered in such oral dosage forms as tablets, capsules (each of

which includes sustained release or timed release formulations), pills, powders,

granules, elixirs, tinctures, suspensions, syrups and emulsions.

The dosage regimen utilising the compounds of the present invention is

selected in accordance with a variety of factors including type, species, age, weight,

sex and medical condition of the patient; the severity of the condition to be treated;

the route of administration; and the particular compound employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective

amount of the compound required to prevent, counter or arrest the progress of the condition

The adult dosage may, for oral application, range from 0.001 to 5g daily,

more preferably 0.0 lg to 0.5g daily.

The preferred daily dose is from 0.000014g to 0.0715g per kg of body weight,

and more preferably from 0.00014g to 0.00715g. Thus for a 70kg adult the daily

dose can range from 0.00 lg to 5.0g, more preferably from 0.01 to 0.5g. This daily

dose may be administered in divided doses from 1 to 4 times a day giving unit doses

for an adult of from 0.00025 to 5.0g.

The compounds of the present invention can form the active ingredient, and

are typically administered in admixture with suitable pharmaceutical diluents,

excipients or carriers suitably selected with respect to the intended form of

administration.

For instance, for oral administration in the form of a tablet or capsule, the

active drug component can be combined with an oral, non-toxic, pharmaceutically

acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose,

magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the

like; for oral administration in liquid form, the oral drug components can be

combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as

ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable

binders, lubricants, disintegrating agents and coloring agents can also be incorporated in to the mixture. Suitable binders include starch, gelatin, natural sugars such as

glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia,

tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes

and the like. Lubricants used in these dosage forms include sodium oleate, sodium

stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and

the like. Disintegrators include, without limitation, starch, methyl cellulose, agar,

bentonite, xanthan gum and the like.

The compounds may be administered by any means that treat and/or prevent

conditions where full or partial inhibition of bone resoφtion is desirable. Such

conditions include osteopenia, which includes osteoporosis, Paget^'s disease, bone

metastases, myeloma, perodontal disease and humeral hypercalcaemia or malignancy.

Compounds of the present invention may be useful for treating and/or

preventing conditions prevalent in post-menopausal women; in particular those

individuals suffering from osteoporosis.

Furthermore, the compounds of the present invention may be useful for

treating and/or preventing conditions prevalent in Caucasian elderly men (e.g.

individuals over 50 years of age); in particular, those individuals suffering from

Paget's disease.

In addition, the compounds of the present invention may be useful in

preventing accelerated bone loss in individuals who are genetically disposed to suffer

from disease where loss of bone occurs.

According to another aspect of the present invention there is provided a composition which will act as an inhibitor or antagonist of an ABC protein for use

in the manufacture of a medicament for use in the treatment of a disease where full

or partial inhibition of bone resoφtion will result in an improvement in the disease

comprising a compound as described hereinabove and parathyroid hormone (PTH).

The compounds may be administered concomitantly with PTH.

Alternatively, PTH may be administered separately. The mode of administration

may be the same or different as that for the compounds of the present invention.

The dosage of PTH may vary according to the criterion set out for the

compounds of the present invention, as hereinabove described.

The adult dosage may, for oral application, range from 1 to 200 International

Units daily, more preferably 50 to 100 International Units.

The preferred daily dose of PTH, is from 0.014 to 2.9 International Units per

kg of body weight and more preferably from 0.7 to 1.4 International Units. Thus for

a 70 kg adult, the daily dose can range from 1 to 200 International Units, more

preferably 50-100 International Units. This daily dose may be administered in

divided doses from 1 to 4 times a day giving unit doses for an adult of from 12.5 to

200 International Units.

Animals which may be treated according to the methods of the present

invention include all animals which may benefit therefrom. Included in such animals

are humans and horses, although the invention is not intended to be so limited.

This discovery also allows candidate compounds to be screened.

According to a further aspect of the present invention there is provided a method of screening for a compound which will lead to full or partial inhibition of

bone resoφtion, which comprises determining whether the compound acts as an

inhibitor or antagonist of the expression or function of an ABC protein in bone.

According to a further aspect of the present invention there is provided a

method of screening for a compound which will act as an inhibitor or antagonist of

the expression or function of an ABC protein in bone comprising determining

whether the presence of said compound leads to full or partial inhibition of bone

resoφtion.

The method may comprise the use of osteoclasts and/or osteoclast precursors.

Osteoclasts may be obtained from any suitable source. Preferably, human bone,

human bone marrow, human blood or any suitable tissues from experimental animals.

More preferably, osteoclast precursors are obtained from human blood.

According to a further aspect of the present invention there is provided a

method of treatment of a disease where full or partial inhibition of bone resoφtion

will result in an improvement in the disease comprising administering a compound

which will act as an inhibitor or antagonist of an ABC protein in bone.

The invention will be further described, by way of example only, with

reference to the following test data.

Identification of novel ABC transporters from human giant cell tumour

Monoclonal antibodies were raised against cells from human bone. One

antibody was shown to strongly stain the osteoclast. In order to identify the antigen a human bone cDNA expression library constructed in lambda gtl l was

immunoscreened. Two clones were identified of size 300bp and 435bp. A second

separate immunoscreen identified the 435bp clone which was then sequenced and

identified as a partial length cDNA clone encoding a novel ATP binding cassette

(ABC) protein. Subsequently this clone was used to hybridization screen an

osteoclastoma cDNA library in lambda gtl l and 19 clones were identified and

purified. The longest sequenced was 1.5kb and found to be a highly homologous, but

distinct from the 435bp clone and encoded a second novel ABC transporter. Further

sequencing of the additional clones suggests that there may be additional family

members. Comparative sequence analysis and phylogenetic analysis demonstrates

that these ABC's comprise a novel sub family of transporters which have not

previously been described. This novel sub family of ABC transporters would appear

to have restricted tissue expression.

Inhibition of resoφtion bv Glibenclamide.

In order to determine whether ABC proteins were involved in bone resoφtion

the applicant conducted in vitro bone resoφtion assays (as per C.A. Walsh et al,

Journal of Bone and Mineral Research, volume 6, number 7, 1991 with avian

osteoclasts, and Methods in Molecular Medicine, Human cell culture protocols, ed

G.E. Jones, Humana Press, pages 263- 275 with human osteoclasts) to look at the

effect of a well characterised ABC protein inhibitor, Glibenclamide, using avian and

human osteoclasts. Active factors known to have an effect on bone resoφtion and

/or Glibenclamide were also introduced into the assays. The active factors included parathyroid hormone (PTH), which is known to stimulate bone resoφtion in this

system, and ATP since it has been suggested that Glibenclamide inhibits ATP release

from cells (E.M. Schwiebert et al, Cell, Vol 81, 1063-1073, 1995).

The results are shown in Figs. 1 to 3. Fig 1 shows inhibition of resoφtion by

Glibenclamide, enhanced inhibition of resoφtion by Glibenclamide and PTH, and

inhibition of resoφtion by Glibenclamide in the presence of ATP. using avian

osteoclasts in vitro according to the above test;

Fig.2 shows inhibition of resoφtion by Glibenclamide using human

osteoclasts derived from a giant cell tumor according to the above test;

Fig. 3 shows the dose response as inhibition of resoφtion by Glibenclamide

using human osteoclasts according to the above test.

The vehicle is tissue culture medium containing 0.1% dimethyl sulphoxide.

In summary, Glibenclamide at concentrations of 100 micromolar inhibited

resoφtion by settled suspensions of avian bone cells (Fig.1). Inhibition of resoφtion

was enhanced by PTH in the presence of Glibenclamide (Fig. l). In addition,

exogenous ATP does not overcome resoφtion inhibition by Glibenclamide (Fig.l).

These findings were supported using a suspension of giant cells obtained from

a human osteoclastoma or giant cell tumour (Fig.2). Glibenclamide at concentrations

lower than 50 micromolar did not inhibit resoφtion, however, at concentrations of

50 and 100 micromolar resoφtion was depressed (Fig.3). This was confirmed using

a different giant cell population. This data demonstrates that inhibiting ABC

proteins inhibits bone resoφtion. More details of the procedure followed are given below.

Avian osteoclasts were isolated from the femora and tibiae of pre -hatch chicks

and seeded on to sterile devitalised dentine wafer. Cells were allowed to settle for

24 hours after which time the wafers were washed to remove non-adherent cells.

Fresh medium containing active factors (PTH, ATP and/or Glibenclamide) were

added for 72 hours. At the end of this period the wafers were fixed.

Dentine wafers were then washed in PBS at 37°C, fixed in 4% glutaraldehyde

in 0.2% sodium cacodylate, and stained for 5 mins in 1% (w/v) toluidine blue in

0.5% disodium tetraborate. Resoφtion lacunae present on stained devitalised bone

wafers were identified using an Olympus BH2 microscope fitted for incident light

microscopy with metallurgic objectives. The plan area of resoφtion was determined

by point counting using a 10X objective and drawing tube and expressed as a

percentage of the total plan area of the bone wafers.

Human osteoclasts were dislodged from human giant cell tumour (GCT) by

agitation in αMEM. Sterile devitalised dentine wafers were placed in a culture dish

and the GCT suspension dripped over them using a sterile 10ml syringe. The culture

was then incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ for

20 min. Wafers were then removed and washed in PBS to dislodge any non-

adherent cells. Wafers were then transferred to 24 well plates, each containing 900 μl

of αMEM supplemented with 10% foetal calf serum and incubated at 37°C in a

humidified atmosphere of 95% air and 5% CO₂ for 24 hours. Cells were treated by

adding lOOμl of lOx concentration of inhibitor and incubated at 37°C for a further 72 hours as described above. Dentine wafers were then washed, fixed, stained and

viewed as detailed in the avian osteoclast procedure.

Claims

1. A compound which will act as an inhibitor or antagonist of an ABC protein in bone for use in the manufacture of a medicament for use in the treatment

of a disease where full or partial inhibition of bone resoφtion will result in an

improvement in the disease.

2. A compound as claimed in claim 1 selected from the group consisting of

glibenclamide, tolbutamide, chloφropamide, tolozamide, glipizide, gliquidone and

gliclazide.

3. A compound as claimed in claim 1 or 2, wherein the compound is in a

form for oral or intravenous administration.

4. A compound as claimed in claim 1, 2 or 3 which is in a unit dosage form.

5. A compound as claimed in claim 4 wherein the compound is present in unit

dosage form in an amount of from 0.00 lg to 5g.

6. A compound as claimed in claim 4 wherein the compound is present in

unit dosage form in an amount of 0.01 g to 0.5g daily.

7. A compound as claimed in claim 4 wherein the compound is present in unit

dosage form in an amount of from 0.000014g to 0.0715g per kg of body weight

daily.

8. A compound as claimed in claim 4 wherein the compound is present in

unit dosage form in an amount of 0.00014g to 0.00715g per kg of body weight daily.

9. A compound as claimed in any of claims 1 to 8 for use in the manufacture of a medicament for use in the treatment of osteoporosis.

10. A composition which will act as an inhibitor or antagonist of an ABC

protein in bone for use in the manufacture of a medicament for use in the treatment

of a disease where full or partial bone resoφtion will result in an improvement in the

disease comprising a compound as claimed in any preceding claim and parathyroid

hormone.

1 1. A composition as claimed in claim 10 wherein parathyroid hormone is

in a form for oral, subcutaneously or intravenous administration.

12. A composition as claimed in claim 10 or 1 1 which is in a unit dosage

form.

13. A composition as claimed in claim 12 wherein parathyroid hormone is

present in unit dosage form of from 1 - 200 International Units.

14. A composition as claimed in claim 12 wherein parathyroid hormone is

present in unit dosage form of from 50 - 100 International Units.

15. A composition as claimed in claim 12 wherein parathyroid hormone is

present in unit dosage form of from 0.014 to 2.9 International Units per kg of body

weight.

16. A composition as claimed in claim 12 wherein parathyroid hormone is

present in unit dosage form in an amount from 0.7 to 1.4 International Units per kg

of body weight.

17. A method of screening for a compound which will act as an inhibitor or

antagonist of the expression or function of an ABC protein in bone comprising determining whether the presence of said compound leads to full or partial inhibition of bone resoφtion.

18. A method of screening for a compound which will lead to full or partial

inhibition of bone resoφtion, which comprises determining whether the compound

acts as an inhibitor or antagonist of the expression or function of an ABC protein in

bone.

19. A method of treatment of a disease where full or partial inhibition of bone

resorption will result in an improvement in the disease comprising administering a

compound which will act as an inhibitor or antagonist of an ABC protein in bone.

20. A method of treatment of a disease where full or partial inhibition of bone

resoφtion will result in an improvement in the disease comprising administering a

composition comprising a compound, which will act as an inhibitor or antagonist of

an ABC protein in bone, and parathyroid hormone.