JP2003501350A - Pharmaceutical compounds - Google Patents

Abstract

(57) Abstract: Use of a compound that acts as an inhibitor or antagonist of ABC protein expression or function, and a method of screening for a compound that acts as an inhibitor or antagonist of ABC protein expression or function.

Description

Detailed Description of the Invention

The present invention relates to the use of compounds and compositions which act as inhibitors or antagonists of the expression or function of ABC proteins in bone, in particular osteoclast associated ABC proteins, in particular ABC proteins, In particular, compounds and compositions that act as inhibitors or antagonists of the expression or function of osteoclast-associated ABC proteins are used for the treatment of diseases in which complete or partial inhibition of bone resorption leads to amelioration of the disease. It relates to its use in the manufacture of a medicament. Such diseases include, but are not limited to, osteopenia such as osteoporosis, Paget's disease, bone metastases, myeloma, periodontal disease and malignant hypercalcemia.

[0002] The invention also includes determining whether the presence of the above compounds leads to complete or partial inhibition of bone resorption, ABC proteins in bone, particularly osteoclast-associated proteins. It also relates to a method of screening for compounds and compositions that act as inhibitors or antagonists of ABC protein expression or function.

ABC proteins (ATP-binding cassette proteins), also called transport ATPases, are a superfamily of transmembrane proteins involved in the movement of substrates across cell membranes. The ABC protein is abundant in prokaryotes, represents approximately 5% of the entire genome, and exhibits specificity for a diverse range of substrates ranging from peptides and amino acids to ions and sugars. The ABC protein is characterized by the presence of two peptide motifs, Walker A and Walker B motifs. These motifs are common to many nucleotide binding proteins. However, the ABC protein is distinguished from these other proteins by the presence of a third C-sign motif that separates the Walker A and B motifs by a conserved interval.

The importance of ABC proteins in the mammalian system is now being recognized. Several members of the ABC family have been shown to be important in human disease, and these dysfunctions include diversity including cystic fibrosis, multidrug resistance of tumor cells, non-insulin dependent diabetes and adrenoleukodystrophy. Cause various pathological conditions.
These ABC proteins are known to be involved in the translocation of ions and hydrophobic drugs across the plasma membrane. Also, as in the case of the MRP subfamily, other human ABC proteins have been shown to be involved in peptide translocation (PAB) across the tubular linear membrane and phospholipid transport. In addition, human ABC-1 has recently been implicated as a regulator of phospholipid balance and non-classical (signal-independent) secretion of IL1-β in macrophages.

The Applicant has discovered a tissue rich in ABC transporter proteins and osteoclasts in bone,
Immunocytochemistry and hybridisation screening to detect giant cell tumor of bone cD
We identified several novel members of the ABC protein family from NA and human bone cDNA libraries.

This discovery of the Applicant shows that compounds that inhibit or promote the expression or function of ABC proteins, particularly osteoclast associated ABC proteins, are useful in treating conditions resulting from osteoclast function. It was

This role of ABC proteins, especially osteoclast-associated ABC proteins, has not previously been identified.

However, it was discovered that P-glycoprotein is present in osteoblasts (Calcified Tissue International, 1996).
, 58, 3, 186-191) In view of the fact, it can be hypothesized that members of the ABC family of proteins are involved in bone formation.

[0009] More generally, ABC cassette proteins have been implicated in numerous cellular functions. Thus, the osteoclast-related family of ABC transporters regulate many processes within these cells.

The ability of members of the ABC superfamily to regulate volume-activated channels by ATP release has been described in other cell types.

Osteoclasts are cells that are terminally differentiated and are therefore programmed to die by apoptosis. ABC1 members of the ABC superfamily have been implicated in the recognition of apoptotic cells by macrophages, a process believed to involve the transmembrane flux of phosphatidylserine.

The putative inhibition or promotion of certain functions of the ABC protein expressed in bone and osteoclasts has been shown to have a complex effect on bone resorption, eg from inhibition, no effect to stimulation. . Inhibition of osteoclast apoptosis leads to a subsequent elevation and enhanced resorption in functional osteoclast pools. Similarly,
Sulfonylurea sensitivity is conferred on the K / ATP channel by the presence of the ABC transporter, whose inhibition blocks potassium ion efflux and consequent calcium ion influx, thereby promoting insulin secretion. Therefore, blocking osteoclast-associated ABC transporters associated with ion channels is shown to enhance the release of factors that stimulate resorption, including regulatory factors, protons and proteases including cathepsin K. Conversely, the process of osteoclast fusion from mononuclear precursors and the process of cell adhesion, if blocked, leads to reduced resorption. As suggested earlier, cell adhesion is promoted by annexin-mediated binding between phospholipids and extracellular matrix, while translocation of transmembrane phospholipids by the ABC1 transporter
Provides a mechanism for osteoclast fusion.

Applicants have confidently demonstrated that inhibition of the ABC protein using the known inhibitor glibenclamide of the known ABC protein in osteoclasts, including the population, inhibits resorption. These results indicate that ABC protein inhibitors or antagonists are useful therapeutic agents in the treatment of diseases in which inhibition of resorption is desired. These include osteopenia, including osteoporosis, Paget's disease, bone metastases, myeloma, periodontal disease and malignant calcemia.

In addition, Applicants have surprisingly found that when present with a compound of the present invention, a known stimulator of bone resorption, parathyroid hormone (PTH), enhances the inhibitory effect of this compound. I found that According to a first aspect of the present invention, an ABC protein in bone for use in the manufacture of a medicament for the treatment of a disease in which complete or partial inhibition of bone resorption leads to amelioration of the disease. Compounds that act as inhibitors or antagonists of

Examples of existing compounds that have an action similar to the sulfonylurea glibenclamide are: tolubutamide, chloropropamide, tolozamide, glipizide, gliquidone. , And gliclazide.

The compound may be administered orally, intravenously, subcutaneously, or by any other conventional route. For oral use, tablets, capsules (each of which includes a sustained release formulation or a timed release formulation), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions The compounds of the invention can be administered in the form.

Dosing regimens using the compounds of the present invention include the type, species, age, weight, sex and condition of the patient; the severity of the condition being treated; the route of administration; and the particular compound used. It is selected by various factors. One of ordinary skill in the art can readily determine and prescribe the effective amount of the compound required to prevent, counter or arrest the progress of the condition.

This adult dose may range from 0.001 to 5 g per day for oral use, more preferably 0.01 g to 0.5 g per day.

A preferred daily dose is 0.000014 g to 0.0715 g / kg body weight.
, And more preferably 0.00014 g to 0.00715 g. in this way,
For a 70 kg adult, the daily dosage may range from 0.001 g to 5.0 g, more preferably 0.01 to 0.5 g. 0.0 for adults
In divided doses 1 to 4 times a day, equivalent to a unit dose of 0025 to 5.0 g,
This daily dose may be administered.

The compounds of the present invention are capable of forming the active ingredient and are usually administered in admixture with a suitable pharmaceutical diluent, excipient or carrier suitably selected with regard to the intended form of administration.

For example, for oral administration in the form of tablets or capsules, the active drug component may be lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc. For oral administration in liquid form, which may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier, the oral drug ingredient may be any oral, non-toxic, such as ethanol, glycerol, water. It can be combined with a toxic, pharmaceutically acceptable inert carrier. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be included in the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural or 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, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The compound may be administered by any means that treats and / or prevents a condition in which a complete or partial inhibition of bone resorption is desired. Such conditions include osteopenia, including osteoporosis, Paget's disease, bone metastases, myeloma, periodontal disease and malignant hypercalcemia.

The compounds of the present invention are useful for treating and / or preventing a prevalent condition in post-menopausal women, especially those suffering from osteoporosis.

In addition, the compounds of the present invention may be used in caucasian elderly men (eg, those over the age of 50),
In particular, it is useful in treating and / or preventing the symptoms prevalent in people suffering from Paget's disease.

In addition, the compounds of the present invention are useful for preventing accelerated bone loss in persons who are genetically afflicted with a disease in which bone loss occurs.

According to another aspect of the invention, it is used to produce a medicament for the treatment of a disease, wherein the complete or partial inhibition of bone resorption leads to the amelioration of the disease,
A composition that acts as an inhibitor or antagonist of ABC protein in bone, comprising:
There is provided a compound comprising a compound as described above and parathyroid hormone (PTH).

The compound may be administered concomitantly with PTH. Alternatively, PTH may be administered separately. The mode of administration may be the same or different for the compounds of the invention.

The dose of PTH may vary according to the criteria set out for the compounds of the invention as described above.

The adult dose may range from 1 to 200 international units per day, more preferably 50 to 100 international units for oral use.

A preferred daily dose of PTH is 0.014 to 2.9 international units per kg body weight, more preferably 0.7 to 1.4 international units. Thus, for a 70 kg adult, the daily dose would be 1 to 200 international units, more preferably 50-1.
It can range from 00 international units. This daily dose may be administered to adults in divided doses 1 to 4 times per day, equivalent to a unit dose of 12.5 to 200 international units.

Animals that may be treated by the methods of the present invention include all animals that benefit from this. Such animals include humans and horses, but the invention is not intended to be so limited.

This discovery also makes it possible to screen candidate compounds.

According to a further aspect of the invention, complete bone resorption comprising determining whether this compound acts as an inhibitor or antagonist of the expression or function of ABC protein in bone. , Or a method of searching for a compound that causes partial inhibition is provided.

According to a further aspect of the invention, the expression of ABC protein in bone or comprising determining whether the presence of said compound leads to a complete or partial inhibition of bone resorption. Methods of screening for compounds that act as inhibitors or antagonists of function are provided.

The method may comprise the use of osteoclasts and / or osteoclast precursors. Osteoclasts may be obtained from any suitable source. The osteoclast precursors are preferably obtained from human bone, human bone marrow, human blood or any suitable tissue of a laboratory animal, more preferably human blood.

According to a further aspect of the invention, there is a complete or partial inhibition of bone resorption comprising administering a compound that acts as an inhibitor or antagonist of ABC protein in bone. Methods of treating diseases that result in improvement of the disease are provided.

[0037]   The invention will now be further described, by way of example only, with reference to test data.

Identification of a Novel ABC Transporter from Human Giant Cell Tumors Monoclonal antibodies were generated against human bone cells. One antibody was shown to strongly stain osteoclasts. To identify the antigen, a human bone cDNA expression library constructed at λgt11 was immunoscreened. Size 300bp
And two 435 bp clones were identified. A 435 bp clone was identified by a second separate immunoscreen. It was sequenced and identified as a partial length cDNA clone encoding a novel ATP binding cassette (ABC) protein. Subsequently, using this clone, a giant cell tumor of λgt11, cdN
The A library was hybridization screened and 19 clones were identified and purified. The longest sequenced was 1.5 kb, 435 bp
It was found to be highly homologous to the clone but distinct, and the second novel AB
The C transporter was encoded. Further sequencing of the additional clones suggests that additional family members are present. Comparative sequence analysis and phylogenetic analysis indicate that these ABCs comprise a novel subfamily of transporters not previously described. This novel subfamily of ABC transporters appears to have restricted tissue expression.

Inhibition of Resorption by Glibenclamide To determine whether the ABC protein is involved in bone resorption, Applicants used an in vitro bone resorption assay (CA, Wals, on osteoclasts of birds, Wals.
h et al., Journal of Bone and Mineral Research
h, 6, 7, 1991, and on human osteoclasts, Methods i
n Molecular Medicine, Human cell culture
e protocols, G.M. E. Edited by Jones, Humana Press, 2
63-275) to investigate the effect of the clearly characterized ABC protein inhibitor glibenclamide on avian and human osteoclasts.
Active agents known to affect bone resorption and / or glibenclamide were also incorporated into this assay. This activator was suggested to inhibit parathyroid hormone (PTH) and glibenclamide, which are known to stimulate bone resorption in this system, to inhibit ATP release from cells (EM. Schwieb
ert et al., Cell, 81, 1063-1073, 1995) ATP.

[0040]   The results are shown in FIGS.

Vehicle is a tissue culture medium containing 0.1% dimethyl sulfoxide.

In summary, glibenclamide at a concentration of 100 micromolar inhibited resorption by sedimented suspensions of avian bone cells (FIG. 1). Inhibition of resorption was enhanced by PTH in the presence of glibenclamide (Fig. 1). In addition, exogenous ATP
It does not overwhelm the inhibition of reabsorption by glibenclamide (Fig. 1).

These findings were supported with giant cell suspensions of giant cells of human bone or giant cell tumors (FIG. 2). Glibenclamide at concentrations below 50 micromolar did not inhibit resorption, but at concentrations of 50 and 100 micromolar suppressed resorption (Figure 3). This was confirmed using different giant cell populations.
This data shows that inhibiting ABC protein inhibits bone resorption.

[0044]   Further details of the procedure followed are given below.

Avian osteoclasts were isolated from prehatch chick femurs and tibias and seeded on sterile, deactivated dentin wafers. Cells were allowed to settle for 24 hours, after which the wafer was washed to remove non-adherent cells. Fresh medium containing active factors (PTH, ATP and / or glibenclamide) was added after 72 hours. At the end of this period, the wafer was fixed.

The dentin wafers were then washed in PBS at 37 ° C., fixed in 4% glutaraldehyde in 0.2% sodium cacodylate and 1% in 0.5% disodium tetraborate. Stained in% (w / v) toluidine blue for 5 minutes. The lacunae present on the stained deactivated bone wafer was identified using an Olympus BH2 microscope adapted for episcopic microscopy with a metallurgical objective. 10X
The area of the resorption top view was determined by point counting using an objective lens and a drawing tube and expressed as a percentage of the total top view area of the bone wafer.

Human osteoclasts were removed from human giant cell tumors (GCT) by agitation in αMEM. A sterile, deactivated dentin wafer was placed in a culture dish and the GCT suspension was instilled onto it using a sterile 10 ml syringe. The culture was then incubated for 20 minutes at 37 ° C. in a humidified atmosphere of 95% air and 5% CO ₂ . next,
The wafer was removed and washed in PBS to remove any non-adherent cells.
The wafers were then each supplemented with 900 μl αM supplemented with 10% fetal bovine serum.
It was transferred to a 24-well plate containing EM and incubated at 37 ° C. for 24 hours in a humidified atmosphere of 95% air and 5% CO ₂ . Cells were treated by adding 100 μl of 10 × concentration of inhibitor and incubated at 37 ° C. for a further 72 hours as described above. The dentin wafers were then washed, fixed, stained and examined microscopically as detailed in the method for avian osteoclasts.

[Brief description of drawings]

FIG. 1 Inhibition of resorption by glibenclamide, increased inhibition of resorption by glibenclamide and PTH and avian osteoclasts in vitro according to the above studies, and A
Inhibition of reabsorption by glibenclamide in the presence of TP.

FIG. 2 shows inhibition of resorption by glibenclamide using human osteoclasts derived from giant cell tumors according to the above test.

FIG. 3 shows the dose response as an inhibition of glibenclamide resorption using human osteoclasts according to the above study.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 A61P 43/00 121 121 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33 / 566 33/566 A61K 37/24 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, A, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, 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, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Bowler, Wayne Barry England, El 125 C-Liverpool, West Derby, Tow Low 52 (72) Inventor Wagstaff, Simon Christopher England, El357 Prescott , Ecculus Gardens, Honeybone Drive 27 F-term (reference) 2G045 AA40 BA11 BA14 BB20 BB24 CA25 CB01 CB13 DA36 FA16 FB03 4C084 AA02 AA19 DB32 MA52 MA66 NA05 ZA96 ZA97 ZC42 ZC75 4C086 AA01 AA02 MA02 MA04 MA22 DA04 MA22 DA04 MA22 MA04 ZC42 ZC75

Claims (20)

[Claims] 1. An inhibitor or antagonist of ABC protein in bone for use in the manufacture of a medicament for use in the treatment of a disease, wherein complete or partial inhibition of bone resorption leads to amelioration of the disease. A compound that acts. 2. The compound according to claim 1, which is selected from the group consisting of glibenclamide, tolbutamide, chlorpropamide, trozamide, glipizide, gliquidone, and gliclazide. 3. A compound according to claim 1 or 2 wherein the compound is in a form for oral or intravenous administration. 4. A compound according to claim 1, 2 or 3 in the form of a unit dose. 5. A compound according to claim 4, wherein the compound is present in the form of a unit dose in the amount of 0.001 g to 5 g. 6. A compound according to claim 4, wherein the compound is present in unit dosage form in the amount of 0.01 g to 0.5 g per day. 7. A compound according to claim 4, wherein the compound is present in the form of a unit dose in the amount of 0.000014 g to 0.0715 g per kg of body weight per day. 8. The compound contains from 0.00014 g / kg to 0 / kg body weight per day.
． 5. A compound according to claim 4, which is present in the form of a unit dose in the amount of 0715 g. 9. A compound according to any of claims 1 to 8 for use in the manufacture of a medicament for use in the treatment of osteoporosis. 10. Treatment of a disease, comprising a compound according to any one of the preceding claims and parathyroid hormone, wherein complete or partial inhibition of bone resorption results in improvement of the disease. ABC in bone used to manufacture a medicament for use in
Compositions that act as protein inhibitors or antagonists. 11. The composition according to claim 10, wherein the parathyroid hormone is in a form for oral, subcutaneous or intravenous administration. 12. A composition according to claim 10 or 11 in the form of a unit dose. 13. The composition of claim 12, wherein parathyroid hormone is present in a unit dose of 1-200 International Units. 14. The composition of claim 12, wherein parathyroid hormone is present in a unit dose of 50-100 International Units. 15. The composition of claim 12, wherein parathyroid hormone is present in a unit dose of 0.014 to 2.9 international units per kg body weight. 16. The composition of claim 12, wherein parathyroid hormone is present in a unit dose of 0.7 to 1.4 international units per kg body weight. 17. As an inhibitor or antagonist of the expression or function of ABC protein in bone, comprising determining whether the presence of said compound leads to a complete or partial inhibition of bone resorption. A method of searching for a compound that acts by screening. 18. Complete or partial inhibition of bone resorption, comprising determining whether this compound acts as an inhibitor or antagonist of the expression or function of ABC protein in bone. A method of searching for compounds by screening. 19. A disease in which complete or partial inhibition of bone resorption leads to amelioration of the disease, comprising administering a compound that acts as an inhibitor or antagonist of ABC protein in bone. How to treat. 20. Complete or partial bone resorption comprising administering a composition comprising a compound that acts as an inhibitor or antagonist of ABC protein in bone and parathyroid hormone. A method of treating a disease, wherein inhibition results in improvement of the disease.