WO1998037100B1

WO1998037100B1 - Therapeutic use of the smr1 protein and active derivatives thereof

Info

Publication number: WO1998037100B1
Application number: PCT/EP1998/000956
Authority: WO
Filing date: 1998-02-19
Publication date: 1999-03-04

Abstract

The present invention pertains to the use of a peptide molecule consisting in a maturation product of SMR1 (Submandibular rat protein 1) of structural formula QHNPR, as well as the biologically active derivatives of the said peptide, for preventing or treating diseases associated with a mineral ion imbalance in a human or an animal body. More particularly, the present invention relates to the therapeutic use of the above-cited molecules for preventing or treating an hydromineral imbalance in organs and tissues such as kidney, bone, dental enamel, dental ivory, gut matrix, pancreas or glandular gastric mucosa. This invention also deals with therapeutic compositions comprising a pharmaceutically active amount of the above-described therapeutic molecules as well as with therapeutic methods using the said therapeutic compositions. Finally, the present invention relates to processes for selecting ligand molecules that possess an agonist or an antagonist biological activity on the target receptor of the QHNPR pentapeptide as well as to the selected ligand molecules themselves.

Claims

88

AMENDED CLAIMS

[received by the International Bureau on 19 January 1999 (19 01 99), original claims 1-45 replaced by amended claims 1-50 (9 pages)]

1 A therapeutic method for preventing or treating diseases caused by a metabolic imbalance, such as a mineral ion imbalance in a mammal said method comprising administering to the mammal a composition comprising a pharmaceutically active amount of an SMR1 protein, or a maturation product of the SMR1 protein or of one of its biologically active derivatives 2 A therapeutic method for preventing or treating a disorder affecting the bone in a mammal, said method comprising administering to the mammal a composition comprising a pharmaceutically active amount of an SMR1 protein, or a maturation product of the SMR1 protein or of one of its biologically

active derivatives 3 A therapeutic method according to claim 2 for preventing or treating osteoporosis

4 The therapeutic method according to claim 1 or 2 nerein said mammal is a human

5 The therapeutic method according to claim 1 or 2 wherein the therapeutic composition comprises a peptide of formula XQHNPR, wherein X is a hydrogen atom, an ammo acid V, or a polypeptide VR, VRG, VRGP VRGPR or VRGPRR, or one of its biologically active derivatives

6 The therapeutic method according to claim 5 wherein the peptide

XQHNPR or one of its biologically active derivative peptides comprises one or more ammo acids in the D-form

7 The therapeutic method according to claim 1 or 2 wherein the 89

therapeutic composition is a liquid solution

8 The therapeutic method according to claim 1 or 2 wherein the

therapeutic composition is a gel

9 The therapeutic method according to claim 1 or 2 wherein the

therapeutic composition is a dry powder

10 The therapeutic method according to claim 1 or 2 wherein the

therapeutic composition is a controlled drug delivery device

11 The therapeutic method according to claim 1 or 2 wherein the

therapeutic composition is administered locally near a site to be treated 12 The therapeutic method according to claim 1 or 2 wherein the therapeutic composition is administered by the orally route

13 A method for screening ligand molecules that specifically bind to a target receptor for an XQHNPR peptide, comprising the steps of a) preparing a confluent target cell culture monolayer or preparing a target organ specimen or a tissue sample, b) adding a candidate molecule to be tested in competition with a half-saturating concentration of labeled peptide,

c) incubating the cell culture, organ specimen or tissue sample of

step a) in the presence of the labeled candidate molecule for a time sufficient for specific binding to take place, and

d) quantifying the label specifically bound to the target cell culture, organ specimen or tissue sample

14 A method for determining an affinity of ligand molecules that

specifically bind to a target receptor for a XQHNPR peptide, comprising the steps of

a) preparing a confluent target cell culture monolayer or preparing a target organ specimen or a tissue sample, b) adding a candidate molecule to be tested that has been previously

labeled with a radioactive or a nonradioactive label, c) incubating the cell culture, organ specimen or tissue sample of step a) in the presence of the labeled candidate molecule for a time sufficient for

specific binding to take place, d) quantifying the label specifically bound to the target cell culture,

organ specimen or tissue sample

15 A method for screening candidate ligand molecules that possess

an agonist or an antagonist biological activity on a target receptor of an XQHNPR peptide, comprising the steps of a) preparing a biological material comprising a confluent target cell culture monolayer, a target organ specimen or a tissue sample in cryosections or slices,

b) incubating the cell culture, organ specimen or tissue sample of step a) in the presence of (i) 10^"10 - 10^'5 M of a candidate molecule and (n) a

submaximal concentration of QHNPR for a time sufficient for an adeπylate cyclase activation to take place, and

c) quantifying adenylate cyclase activity present in the biological material of step a), respectively in the presence or in the absence of the candidate

ligand molecule and in the presence or in the absence of a submaximal concentration of QHNPR

16 A biologically active derivative of the XQHNPR polypeptide which has been obtained according to the method of claim 15, provided that said bⁱologⁱcally active derivative does not have the following structure Y-HNP-Z, 91

wherein Y denotes a glutamme (Q), a pyroglutamic acid residue, or a sequence of two aminoacids arginine-glutamine (RQ) and Z represents an OH group or a basic

ammo acid, the basic ammo acid being a Lysme (K) or an Argmine (R) or said biologically active derivative does not have the sequence QHNLR or

RQHNLR

17 A method for determining the amount of XQHNPR or one of its biologically active derivatives to be administered to a patient suffering from a metabolic imbalance such as a mineral ion imbalance, comprising the steps of a) incubating a labeled XQHNPR peptide with a polyclonal or a

monoclonal antibody directed against the same peptide, b) bringing into contact immune complexes formed with a biological sample from a patient to be tested suspected to contain an endogenous non-labeled XQHNPR peptide, c) detecting monoclonal or polyclonal antibody-bound labeled peptides that have not been displaced by the endogenous non-labelled XQHNPR

peptide contained in the biological sample in order to determine a concentration of said endogenous peptide that is contained in the biological sample,

d) comparing the concentration of the XQHNPR peptide formed at step c) with the concentration of the XQHNPR peptide normally found in a healthy individual, and

e) calculating the amount of a therapeutic composition, comprising a pharmaceutically active amount of an SMR1 protein, or a maturation product of the

SMR1 protein or of one of its biologically active derivatives, necessary in order to supply the defect of the XQHNPR peptide in body fluids and tissues

18 The method according to claim 17 wherein said XQHNPR peptide is QHNPR

19 A composition comprising a pharmaceutically effective amount of

- an SMR1 protein,

- an SMR1 maturation product, or - a biologicaly active derivative of the above, in combination with a pharmaceutically effective amount of a molecule involved in regulation of metabolic balance, such as a mineral ion balance, in the

body

20 The composition according to Claim 19 wherein said SMR1

maturation product is an XQHNPR peptide

21 The composition according to Claim 20, wherein said XQHNPR

peptide is a QHNPR pentapeptide or a polymer thereof

22 The therapeutic composition according to claim 19 wherein the

molecule involved in the regulation of the mineral ion balance in the body is parathyroid hormone (PTH)

23 A composition according to claim 19 wherein the molecule involved in the regulation of the mineral ion balance in the body is calcitonm (CT)

24 A therapeutic composition according to claim 19, wherein the molecule involved in the regulation of the mineral ion balance in the body is 1 ,25-dιhydroxyvιtamιn D

25 A method for producing an SMR1 protein maturation product which comprises the steps of

a) optionally amplifying a nucleic acid coding for a desired polypeptide using a pair of primers specific for an SMR1 genomic or cDNA sequence,

b) inserting a nucleic acid coding for SMR1 protein inserted in an 93

appropriate vector, c) inserting a nucleic acid coding for furin in a suitable vector, said vector being the vector of step b) or said vector being a vector different form the

vector of step b) d) culturing, in an appropriate culture medium devoid of serum, a cell host previously transformed or transfected with the recombmant vector of step b)

and c), e) harvesting the culture medium and the cell host, f) separating or purifying, from said culture medium or from a pellet of a

resultant host cell lysate, the thus produced polypeptide of interest, g) characterizing the produced polypeptide of interest, and h) optionally assaying for specific recognition of said peptide by a polyclonal or a monoclonal antibody directed against a WQHNPR peptide

26 The method according to claim 25, wherein said SMR1 protein maturation product is an XQHNPR peptide

27 The method according to claim 25, wherein said amplifying step a) is carried out by SDA, TAS, 3SR NASBA, TMA, LCR, RCR, CPR, Q-beta replicase or PCR

28 The method according to claim 25, wherein the harvesting step e) is carried out by lysing the cell host by sonication or by an osmotic shock

29 The method according to claim 25 wherein the WQHNPR peptide is a QHNPR peptide

30 A method for screening candidate ligand molecules that possess

an agonist or an antagonist biological activity on a target receptor of an XQHNPR peptide, comprising the steps of

ARTICLE 191 a) culturing a eukaryotic cell capable of synthesizing collagen b) incubating the eukaryotic cell of step a) in beta-glycerophosphate in the presence of 10¹⁰ - 10^'5 M of the candidate molecule and in the presence of a

submaximal concentration of QHNPR peptide, c) quantifying production of a specific metabolite in the presence or in

the absence of the candidate ligand molecule and in the presence or in the absence

of a submaximal concentration of QHNPR

31 The method according to claim 30, wherein said specific

metabolite is calcium, alkaline phosphatase or DNA synthesis 32 A method for screening a candidate ligand molecule that possesses an agonist or an antagonist biological activity on a target receptor of an

XQHNPR pentapeptide, comprising the steps of a) preparing a confluent target cell culture monolayer or preparing a target organ specimen or a tissue sample in cryosections or slices, b) incubating the cell culture, organ specimen or tissue sample of step a) in the presence of 10^"10 - 10^"5 M of the candidate molecule and in the presence of a submaximal concentration of QHNPR,

d) quantifying production of a corresponding metabolite, respectively in the presence or in the absence of the candidate ligand molecule and in the presence or in the absence of a submaximal concentration of QHNPR

33 The use of an SMR1 protein, or a maturation product of the SMR1 protein or of one of its biologically active derivatives for the preparation of a

medicament for preventing or treating diseases caused by a metabolic imbalance, such as a mineral ion imbalance in a mammal

34 The use of an SMR1 protein, or a maturation product of the SMR1 protein or of one of its biologically active derivatives for the preparation of a medicament for preventing or treating a disorder affecting the bone in a mammal

35 The use according to claim 34 for preventing or treating

osteoporosis. 36 The use according to claim 33 or 34 wherein said mammal is a

human.

37 The use according to claim 33 or 34, wherein the medicament

comprises a peptide of formula XQHNPR, wherein X is a hydrogen atom, an ammo acid V, or a polypeptide VR, VRG, VRGP, VRGPR or VRGPRR, or one of its biologically active derivatives.

38 The use according to claim 37 wherein the peptide XQHNPR or one of its biologically active derivative peptides comprises one or more ammo acids

in the D-form

39 The use according to claim 31 or 34 wherein the medicament is a

liquid solution

40 The use according to claim 33 or 34 wherein the medicament is a gel.

41. The use according to claim 33 or 34 wherein the medicament is a dry powder.

42. The use according to claim 33 or 34 wherein the medicament is a controlled drug delivery device.

43. The use according to claim 33 or 34 wherein the medicament is administered locally near a site to be treated.

44. The use according to claim 33 or 34 wherein the medicament is administered by the orally route. 45. A tissuiar receptor complex for the SMR1-pentapeptide of

sequence QHNPR.

46. A tissuiar receptor complex according to claim 45 with a pHi of 5.58/5.64 ± 0.30, mainly present in the medulla of kidney, and pancreas and in

lesser extent in the bone trabecular matrix.

47. A tissuiar receptor complex according to claim 45 with a pHi of 6.62

± 0.35 mainly present in the glandular gastric mucosa.

48. The use of the tissuiar receptor according to claims 45 to 47 for

screening ligands having an affinity for said receptor. 49. The use of the tissuiar receptor according to claim 48 comprising the steps of :

- contacting said tissuiar receptor with a potential ligand under conditions allowing the binding of said potential ligand by said tissuiar receptor; and

- detecting the complex formed thereby. 50. The use of an SMR1 protein or a maturation product of the SMR1 protein or one of its biologically active derivatives for the preparation of a medicament active on renal tubular reabsorption through the complex described in claim 45.