JP2002511271A - Novel membrane metalloprotease NEPII and its use for screening inhibitors useful in therapy - Google Patents

Abstract

  (57) [Summary] The present invention relates to an amino acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 4, a derivative or homologous sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a biologically active fragment of SEQ ID NO: 2 or SEQ ID NO: 4. An isolated polypeptide, referred to as "NEP II," and the use of said NEP II polypeptide for therapeutically useful screening inhibition.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel membrane metalloprotease called NEP II and its use for screening inhibitors particularly useful in therapy.

[0002] Membrane metalloproteases, such as neprilysin (NEP I, EC 3.4.24.11), play an important role in the activation or inactivation of neuronal or hormonal peptide messengers. From their selective inhibition by synthetic compounds, in particular gastrointestinal diseases (Baumer et al., Gut. 1992, 33: 753-758) and cardiovascular (Gros et al., Proc. Natl. Acad. Sci. USA, 199).
1, 88: 4210-4214), drugs that are currently being used in therapy or in clinical development are being led. The isolation of cDNAs of new analogous metalloprotease genes allows the development of a new class of specific inhibitors for promising therapeutic applications. For example, the cloning and expression of the gene for endothelin converting enzyme (ECE) (Xu et al., Cell, 1994, 78: 473-485) has enabled the development of potentially useful inhibitors in certain cardiovascular diseases. .

The inventor of the present application has identified a novel membrane metalloprotease belonging to the ECE / NEP / Kell family, referred to as NEP II (Lee S. et al., 1991,
PNAS 88 (14): 6353-57).

[0004] The invention therefore relates to the sequence of SEQ ID No. 2 or SEQ ID No. 4, the derivation or homologous sequence of such a sequence SEQ ID No. 2 or SEQ ID No. 4, or the biological sequence of the sequence SEQ ID No. 2 or SEQ ID No. 4 The present invention is directed to an isolated polypeptide comprising an amino acid sequence selected from a highly active fragment, and such an isolated polypeptide is referred to as "NEP II."

[0005] The sequence of SEQ ID NO: 2 is the amino acid sequence of NEP II identified in rat.

[0006] The sequence of SEQ ID NO: 4 is the (partial) amino acid sequence of NEP II identified in humans.

An “derived” polypeptide is derived from a genetic and / or chemical modification of the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, ie, a mutation, deletion, addition, substitution and / or chemical modification of at least one amino acid. It refers to all polypeptides resulting from the modification, or all isoforms having a sequence identical to SEQ ID NO: 2 or SEQ ID NO: 4, but comprising at least one D-amino acid.

The above substitutions are preferably conservative substitutions, ie amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonine and tyrosine), amino acids with basic side chains (lysine, arginine) And histidine)
Substitution of amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with non-polar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and cysteine) And substitutions of amino acids of the same class.

“Homologous” polypeptide refers to any polypeptide that can be isolated, especially in mammalian species other than rat or human.

The above homologous polypeptide preferably has a sequence homology of 70% or more, more preferably 75% or more with the complete sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and contains the active site. The homology is particularly high in parts.

[0011] Homology is generally determined by software for sequence analysis (eg, The Genetics, Inc.).
Computer Group's Sequence Analysis Sof
Tare Package, University of Wisconsi
n Biotechnology Center, 1710 Universi
ty Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain maximum homology (ie, identity). This may require artificial insertion of voids ("gaps") in the sequence. Once an optimal alignment is achieved, the degree of homology (ie, identity) is ascertained by recording all positions where the amino acids of the two sequences being compared are identical relative to the total number of positions.

The above-described derived or homologous polypeptide, or a polypeptide fragment of a polypeptide having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, is biologically active, ie, having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. It has the same or similar biological properties as the NEP II polypeptide, ie, metalloprotease activity.

[0013] Preferred polypeptide fragments comprise an active site sequence which is responsible for binding the zinc atom which is essential for catalysis. This active site was identified as containing a HEX ₁ X ₂ H residue. X ₁ and X ₂ represent various amino acids. NEP, especially in rats and humans
HEITH sequence in the II polypeptide (amino acid 60 of the sequence of SEQ ID NO: 2)
8-612).

The present invention also provides an isolated sequence comprising a nucleotide sequence selected from the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, a derivative or homologous sequence of such a sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a complementary sequence thereof. Target nucleic acid.

[0015] The sequence of SEQ ID NO: 1 is a cDNA sequence containing a region encoding NEP II identified in rat.

The sequence of SEQ ID NO: 3 is a cDNA sequence that includes (partially) the region encoding NEP II identified in humans.

An “derived” nucleotide sequence is defined as any nucleotide sequence encoding a derived polypeptide of NEP II as defined above, ie, a modification of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, especially at least one nucleotide Means a sequence generated by mutation, deletion, addition or substitution of In particular, it includes sequences derived from the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 due to the degeneracy of the genetic code.

“Homologous” polypeptides include, in particular, NEP I homologous to a NEP II polypeptide of sequence SEQ ID NO: 2 or SEQ ID NO: 4 in a mammalian species other than rat or human.
All nucleotide sequences encoding an I polypeptide are meant.

Such a homologous sequence preferably comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3
%, More preferably at least 75%, and the homology is particularly high in the central portion of the sequence encoding the NEP II polypeptide.

Preferably, such a homologous nucleotide sequence will specifically hybridize under stringent conditions to a sequence that is complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The parameters defining stringency conditions depend on the temperature (Tm) at which 50% of the duplex separates.

For sequences containing more than 30 bases, Tm is determined by the following formula: Tm
= 81.5 + 0.41 (% G + C) + 16.6 log (cation concentration)-0.63
(% Formamide)-(600 / number of bases) (Sambrook et al., Molecu
lar Cloning, A laboratory manual, Co
ld Spring Harbor Laboratory Press, 19
89, p. 9.54-9.62).

For sequences less than 30 bases in length, Tm is defined by the following equation:
m = 4 (G + C) +2 (A + T).

Under appropriate stringency conditions, where non-specific sequences do not hybridize,
The hybridization temperature is about 5-30 ° C, preferably 5-1 from Tm.
The temperature is 5 ° C., more preferably 5 to 10 ° C. below Tm (high stringency), and the hybridization buffer used is preferably a solution having a high ionic strength such as a 6 × SSC solution.

[0024] The nucleotide sequence according to the present invention can be used for the production of a recombinant NEP II protein according to the present invention according to recombinant product generation techniques known to those skilled in the art.

An effective system for the production of recombinant proteins requires the use of, for example, plasmid or viral derived vectors and compatible host cells.

The cell host may be a prokaryotic cell line such as a bacterium or a prokaryotic cell line such as a mammalian cell such as, for example, yeast, insect cells, CHO cells (Chinese hamster ovary cells), or other cells that may be conveniently used. You can choose from the system.

The vector must contain a promoter, translation initiation and termination signals, and appropriate regions for transcriptional regulation. It must be able to integrate into the cell, and in some cases may have specific signals that direct secretion of the translated protein.

[0028] These various control signals are selected depending on the cell host used. To that end, the nucleotide sequence according to the invention can be inserted into a vector that replicates autonomously in the selected host or into an integration vector of the selected host. Such vectors are produced according to methods routinely used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods such as, for example, electroporation.

Examples of vectors of interest include plasmid pcDNA3.1, PCR2.1 (I
nvitrogen) or pMbac (Staratagene).

The present invention is directed to cloning and / or expression vectors containing a nucleotide sequence according to the present invention, and further to host cells transfected by those vectors. The cells are obtained by introducing a nucleotide sequence inserted into a vector as defined above into a host cell, and then culturing such cells under conditions that allow the transfected nucleotide sequence to replicate and / or express. Obtained by

The cells can be used in a method for producing a recombinant polypeptide according to the invention.

The method for producing a recombinant form of the polypeptide of the present invention comprises culturing the transfected cells under conditions which are themselves encompassed by the present invention and permit the expression of the recombinant polypeptide according to the present invention. And recovering such a recombinant polypeptide.

The purification steps used are known to those skilled in the art. Recombinant polypeptides can be used separately or in combination from lysates of cell supernatants and cell extracts, such as fractionation, chromatography, immunoaffinity techniques using monoclonal antibodies or polyclonal sera, etc. Can be purified by

The present invention also relates to a genomic DNA or a medium encoding a polypeptide according to the present invention.
Strongly and specifically hybridizes with the nucleic acid sequence of
Target nucleotide probes. Appropriate hybridization conditions are
Temperature and ionic strength conditions commonly used by those skilled in the art (Sambrook et al.).
1989), preferably at high stringency conditions, ie (T_m5 ° C
(Below) to (T_m15 ° C. below), more preferably T_mFrom (T _m 10 ° C. below) (high stringency).

Preferred probes are oligonucleotide probes selected in particular from the sequences SEQ ID NO: 5 to SEQ ID NO: 27.

[0036] Such probes are used for specific amplification reactions according to sequencing or so-called PCR (polymerase chain reaction) techniques or other variations of such techniques.

Such a probe can also be used to detect N in a cell or tissue extract or in a cell or tissue by in situ hybridization, including the following steps:
Used in a process for detecting the expression of an EP II polypeptide:-preparing the extract or the RNA of the cell or the tissue;-making the obtained RNA specific for a nucleotide sequence according to the invention. Is contacted with at least one probe having a nucleotide sequence capable of hybridizing to a nucleic acid, provided that such probe can be, in particular, an oligonucleotide probe of the sequence SEQ ID NO: 5 to SEQ ID NO: 27; The presence of an mRNA that hybridizes with the probe, which serves as an indicator, is detected.

The present invention also relates to a monoclonal or polyclonal antibody or a fragment thereof, or a chimeric antibody obtained from a polypeptide of the present invention administered to an animal and capable of specifically recognizing the polypeptide according to the present invention. Alternatively, the target is an immune complex antibody. The present invention is further directed to the use of these antibodies for the purification or detection of NEP II polypeptide in a biological specimen.

Polyclonal antibodies can be obtained, for example, from the sera of animals immunized against the NEP II protein, produced by genetic recombination according to the methods described above using conventional operating modes.

[0040] Monoclonal antibodies are available from Kohler and Milstein (Nature, 1
975, 256, p. 495-497) can be obtained according to the classical method of hybridoma culture described above.

The antibodies may be chimeric antibodies, humanized antibodies, Fab and F (ab ′) 2 fragments.
Antibodies can also take the form of immunoconjugates or labeled antibodies.

[0042] The antibodies according to the invention are used in particular for detecting the presence of NEP II.

The present invention is therefore directed to a method for the immunological detection of NEP II in a cell or tissue extract or in a cell or tissue comprising the following steps: Contacting the tissue extract, the cells or the tissue with an antibody detectable according to the invention; detecting the presence of said antibody, which indicates expression of a NEP II polypeptide.

A “detectable antibody” is an antibody that has been labeled with a detectable group, such as radioactivity, an enzyme, a fluorogene or a fluorescent group, or that is itself detectably labeled. Means an antibody bound to another antibody.

The antibodies according to the invention can therefore be particularly indicative of neuroendocrine tumor cells,
Overexpression of polypeptide II can be assessed.

The present invention is also directed to a method for identifying a substrate compound of the polypeptide NEP II as defined above. In this method, the compound is converted to the polypeptide NEP II
And evaluate cleavage of the optionally labeled compound by NEP II, which is an indicator of the metalloprotease activity of NEP II on the compound.

The substrate specific for NEP II is used in particular for a method for detecting the metalloprotease activity of NEP II in or in a cell or tissue extract, said method comprising the steps of: Contacting the substance or cell or tissue with a substrate compound of the polypeptide obtained and optionally labeled according to the present invention, and evaluating the cleavage of said substrate compound, which is an indicator of the metalloprotease activity of NEP II.

The test cell is in particular a cell transfected with a polypeptide encoding the polypeptide NEP II as defined above. The test tissue extract is, inter alia, the membrane of the testis, which is particularly rich in the metalloprotease NEP II.

In another aspect, the present invention is directed to a method of screening for a compound that inhibits the metalloprotease activity of polypeptide NEP II according to the present invention.
In the method, the compound is contacted with the polypeptide NEP II and
The percent inhibition of PII metalloprotease activity is evaluated.

The compound that inhibits the metalloprotease activity of NEP II is preferably a short peptide consisting of two or three natural or modified amino acids.

The synthetic peptide identified as an inhibitor of the metalloprotease activity of NEP II by the above screening method can be conjugated to a chelating group of zinc such as thiol, phosphate or hydroxamic acid by conventional techniques known to those skilled in the art. it can. The resulting inhibitory compound is combined with a pharmaceutically acceptable vehicle, making it a suitable candidate for a pharmaceutically active ingredient. The chelating groups may optionally be temporarily protected, for example by a thiol ester, to improve the bioavailability of the active ingredient.

The NEP II polypeptides of the present invention are particularly useful for screening for compounds that inhibit the metalloprotease activity of NEP II used to manufacture a medicament for treating a disorder due to peptide transmission involving NEP II. It is.

Among the disorders of interest are cardiovascular diseases, neurodegenerative diseases, endocrine-derived growth disorders, hypothalamic-pituitary nerve modulation and endocrine conditions.

Also, a substrate molecule of NEP II or an inhibitor of the metalloprotease activity of NEP II obtained by the above method may be useful for detecting NEP II protein.

The present invention is also a method for detecting NEP II in a cell extract or tissue extract or in a cell or tissue, the method comprising: labeling the cell extract or tissue extract with the polypeptide NEP II Contacting with a labeled substrate compound or a labeled inhibitory compound of the metalloprotease activity of NEP II as obtained according to the screening method defined above, a substrate compound or inhibitory compound indicative of the presence of the polypeptide NEP II Detecting the presence of

The term “labeled substrate compound” or “labeled inhibitor” refers to a substrate compound or a label that is labeled in a manner detectable by an atomic group, for example, radioactive, enzymatic, fluorescent, fluorescent, etc. Means an inhibitory compound.

Hereinafter, the present invention will be illustrated by way of examples without limitation.

[0058]Example 1: Cloning of cDNA encoding rat NEP II  Align peptide sequences of enzymes ECE, NEP I and Kell
Degenerate oligonucleotides were obtained by delimiting the zones.

Total RNA from various rat tissues (brain, intestine, testis) was reverse transcribed (RT) and amplified by polymerase chain reaction (PCR) using a pair of degenerate oligonucleotides in the cysteine-rich N-terminal region. .

The sequences of these degenerated oligonucleotides are as follows: DCYS2 CCC AAG (G / T) CG (A / G) G (A / G) CTG GTC DCYS3 T (A / T) (C / T) GC (A / C / T / G) GG (A / T) GG (A / C) TGG This allowed the amplification of a 420 bp fragment encoding an open reading frame showing 76% homology with the protein NEPI from testis tRNA. This sequence was completed from brain and testis tRNAs by 3'RACE (rapid amplification of cDNA ends) and 5'RACE. These sequences were confirmed by examination of five different clones for each tissue and each amplification. Then, complete cDN
A (SEQ ID NO: 1) was prepared using the vector PCR2.1 and pcDNA3.1 (Invitrogen).
Cloned.

[0061]Example 2: Characterization of rat polypeptide NEP II  The isolated novel gene encodes a 774 amino acid protein (SEQ ID NO: 2).
With high homology to the enzymes NEP I, ECE and Kell (each
And 52%, 40% and 28% amino acids are identical).
HEXXH, a transmembrane domain following four cysteine residues characteristic of this family
Region (amino acids 24 to 40 of SEQ ID NO: 2) and seven glycosylations are possible
It has a unique part. Three alternatives by RACE sequencing and RT-PCR
Splicing sites were identified. One of these alternative splicing sites
Removes one site where glycosylation is possible, and
Affects cell surface permeation or its activity. Each splicing site is NEP
 I corresponds to the exon of I, suggesting a similar gene structure.
These results indicate that this novel enzyme is a metalloprotease ECE / NEP / Kel
It belongs to the family of l. Extremely high homology with NEP I
This is why it was named NEP II.

[0062]Example 3: Cloning of cDNA encoding human NEP II  To clone human NEP II analogs, rat NEP II
Two oligonucleotides were designed based on the protein sequence. These arrays
Is less degenerate (e.g., represented by a single codon in the genetic code)
Tryptophan) and conservation (eg, zinc binding site)
did.

1- (H) EITHFD (SEQ ID NO: 28) or 5′-CGA GAT CA
C ACA TGG CTT TGA TGA-3 ′ (S) (SEQ ID NO: 22) 2-QVWCGS (SEQ ID NO: 29) or 5′-GGA CCC ACA C
CA CAC CTG-3 '(AS) (SEQ ID NO: 23).

The human hippocampus cDNA obtained from the bank (Stratagene) was subjected to polymerase chain reaction to amplify, subclone and sequence the 330 bp band (SEQ ID NO: 3). The resulting sequence shows 82% sequence homology with rat NEP II, confirming that this sequence encodes a human analog.

The presence of the zinc binding site HEITH was confirmed by 5′RACE using human-specific oligonucleotides HNII-2 and HNII-3. Further, by using the oligonucleotides HNII-1 and HNII-2,
The 3 'region can be amplified by the 3' RACE technique.

HNII-1 5′-CGG CCT GGA TCT CAC CCA TGA G-3 ′ (SEQ ID NO: 24) HNII-2 5′-CTG ACT GCT CCC GGA AGT GCT GGG TG-3 ′ (SEQ ID NO: 25) HNII- 3 5'-GAG CAG CTC TTC TTC ATC-3 '(SEQ ID NO: 26) HNII-4 5'-CTC CAC CAA TCC ATC ATG TTG C-3' (SEQ ID NO: 27).

[0067]Example 4: Tissue expression of NEP II  By Northern blot and RT-PCR studies, NEP II
Very highly expressed in testis, moderately expressed in heart, liver, gastrointestinal tract and brain
2.8 kb transcript. Also semi-quantitative
RT-PCR studies showed similar expression profiles in these tissues as well as
The long chain form is shown to be predominant.

All of these properties clearly show that the protein identified here is one of the membrane metalloproteases (ectoproteases) involved in the metabolism of neuronal or hormonal peptide messengers.

The natural polypeptide NEP II is found in the nervous system, glands (pituitary, testis),
It is heterogeneously expressed in the small intestine, especially in the cardiovascular organs (especially the heart).

Furthermore, in situ hybridization techniques have shown that the protein NEP II is strongly expressed in pituitary cells and neurons that express the gene for the precursor of ACTH, POMC (propiomelanocortin). .

Such localization indicates that NEP II is involved in the proteolysis of hormones and peptidic neurotransmitters or their precursors released from or acting on these various organs. Is shown. Therefore, it is important for therapeutic purposes to inhibit NEP II by acting on the corresponding peptidic transduction.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4C084 1/21 9/64 Z 4H045 5/10 9/99 9/64 C12P 21/08 9/99 C12Q 1/37 C12P 21/08 1/68 A C12Q 1/37 G01N 33/573 1/68 C12N 15/00 ZNAA G01N 33/573 5/00 A (72) Inventor Rose, Kristianu France, F-78320 Le Mesnil-Saint-Deuni, Plas-Henri-Kyatre, 20 (72) Inventor Bonhomme, Mari-Chalantal Canada, Quebec-Aciyu 4 El 1 G 8, Saint-Laurent, La Pointe, 910 (72) Inventor Huacinettei, Patricia F-94420 Le Presi Trevis, France -Dou-Jouene d'Auard, 31 (72) Inventor Shiwvarz, Jijan-Cijar, France, F-75014 Paris, Villa Sura, 9F term (reference) 4B024 AA11 CA04 CA09 GA11 HA12 4B050 CC01 CC03 DD11 LL03 4B063 QA01 QA18 QA19 QQ02 QQ20 QQ52 QR16 QR56 QR57 QS14 QS28 QS34 4B064 AG27 CA10 DA13 4B065 AA01X AA57X AA90X AA93Y AB01 AC14 BA01 CA31 CA46 4C084 AA17 DC32 NA14 ZA011 AA45 ZA76A DA78

Claims (14)

[Claims] An amino acid selected from SEQ ID NO: 2 or SEQ ID NO: 4, a derivative or homologous sequence of such SEQ ID NO: 2 or SEQ ID NO: 4 or a biologically active fragment of such a sequence of SEQ ID NO: 2 or SEQ ID NO: 4 Containing an array, "N
An isolated polypeptide designated "EP II." 2. An isolated nucleic acid comprising a nucleotide sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3, a derivative or homologous sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a complementary sequence thereof. 3. An oligonucleotide probe that specifically hybridizes with the nucleotide sequence according to claim 2, having a nucleotide sequence selected from SEQ ID NOs: 5 to 27. 4. A cloning vector and / or expression vector comprising the nucleotide sequence according to claim 2. A host cell transfected with the vector according to claim 4. 6. A monoclonal or polyclonal antibody or a fragment thereof, which is obtained from the polypeptide of claim 1 and is capable of specifically recognizing the polypeptide of claim 1, which is administered to an animal. Or an immune complex antibody. 7. A method for immunologically detecting NEP II in a cell extract or tissue extract or in a cell or tissue, wherein the cell extract or tissue extract or the cell or tissue is detected according to claim 6. Contacting the antibody with a possible antibody; and assessing the presence of an antibody indicative of the presence of the polypeptide NEP II. 8. A method for detecting the expression of polypeptide NEP II in a cell extract or tissue extract or in a cell or tissue by in situ hybridization, comprising preparing RNA of said extract or cell or tissue. Contacting the obtained RNA with at least one probe having a nucleotide sequence capable of specifically hybridizing with the nucleotide sequence according to claim 2,
4. A method as claimed in claim 3, wherein the probe is in particular an oligonucleotide probe according to claim 3, and the step of assessing the cleavage of the substrate compound which is indicative of the expression of a NEP II polypeptide. 9. The method according to claim 9, wherein the optionally labeled compound is contacted with a polypeptide NEP II, and the cleavage of the compound by NEP II which is an indicator of the metalloprotease activity of NEP II on the substrate compound is evaluated. A method for identifying a substrate compound of the polypeptide NEP II according to claim 1. 10. A method for detecting the metalloprotease activity of NEP II in or in a cell extract or tissue extract, wherein the cell extract or tissue extract or the cell or tissue is according to claim 9. Contacting an optionally labeled polypeptide compound obtained according to the method with a substrate compound; and evaluating the cleavage of the substrate compound that is indicative of NEP II metalloprotease activity. 11. The metalloprotease of the polypeptide NEP II according to claim 1, wherein the compound is brought into contact with the polypeptide NEP II, and the inhibition rate of the metalloprotease activity of NEP II is evaluated. A method for screening a compound capable of inhibiting the activity. 12. A method for detecting the polypeptide NEP II in a cell extract or tissue extract or in a cell or tissue, wherein the substrate compound is a polypeptide compound of the polypeptide NEP II obtained by the method according to claim 9. Contacting the NEP II metalloprotease activity-inhibiting compound obtained by the screening method according to item 11 with the cell extract or tissue extract or the cell or tissue which may be optionally labeled; and Detecting the presence of the substrate compound or the inhibitory compound, which is indicative of the presence of peptide NEP II. 13. The method according to claim 1, for screening a compound that inhibits the metalloprotease activity of NEP II for use in the manufacture of a medicament for treating a disorder associated with peptidic transmission involving NEP II. Peptide NEP
Use of II. 14. The use according to claim 13, wherein the disorder is selected from cardiovascular diseases, neurodegenerative diseases, endocrine-derived growth disorders, hypothalamic-pituitary nerve modulation and endocrine conditions. .