Note: Descriptions are shown in the official language in which they were submitted.
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FRAG~DENTS OF LEPTnN(OB PROTEnN)
The invention relates to novel compounds, in particular to novel peptides, to
compositions cont~ining such compounds and to the use of such compounds in
S medicine.
The mechanism of the physiological regulation of energy balance in the body -
1~ food intake verses energy output - has been the subject of debate for many years. In a
recent publication in Nature Y. Zhang et al, (Nature, 372, 425-431, 1994) suggest that
one of the molecules which plays a key role in energy balance regulation is the ob
protein. Zhang et al also report the cloning and sequencing of both mouse and human
ob gene protein or leptin.
The structure of human leptin or (human ob protein) and its use in the
modulation of body weight in ~nim~ is disclosed in United Kingdom Patent
application Publication Number GB2292382. This application also discloses certain
fr:~gment~ of leptin which are also stated to be capable of modnl~ting body weight.
Collins et al in Nature, Vol 380, page 677, 1996 disclose that the weight
}educing~)Lopel lies of leptin may be accounted for by an enhancement of energy
utilization as well as decreasing food uptake
We have now discovered certain novel fr~gment.~ of leptin which surprisingly
are indicated to modulate body weight ~ul~ y by means of ~nh~nt~in~ energy
utilization. These fragments are therefore considered to be of particular use in the
treatment of nutritional and metabolic disorders, particularly obesity and diabetes.
Accordingly, in a first aspect, the present invention provides a peptide or a
functional derivative, analogue or variant thereof, which modulates body weight,substantially be means of morlnl~ting energy utilisation.
Preferably the modulation of body weight is a reduction of body weight.
Preferably the modulation of energy utilisation is via an enhancement of
energy utilization.
Preferably, the peptide is a fragment of an ob protein, especially human ob
protein, or a functional derivative, analogue or variant thereof.
H~ieillarl~ protein fr~gment~ (or peptides) will be referred to with reference to
the amino acid sequence of human ob protein, using an analogous abbreviation to the
following: 'the protein fragment con~i~ting of amino acid residues 1 to 6' is
abbreviated to 'obl-6'.
Particular peptides include ob21-26 (MVPIQK), ob27-32 (VQDDTK), ob33-
36 (TLIK),ob37-41 CI~VTR), ob42-54 ~NDISHTQSVSSK), obSS-56 (QK), obS7
-74 ~VTGLDFIPGLHPILTLSK), ob93 -105 ~NVIQISNDLENLR), obl06-115
(DLLHVLAFSK), obl16-149
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W~ 97/46S85 PCT~P97/02968
(SCHLPWASGLETLDSLGGVLEASGYSTEVVALSR) and ob l S0- 167
(LQGSLQDMLWQLDLSPGC) especially obS7 -74 ~VTGLDFIPG~HPILTLSK)
Suitably, the invention includes a peptide formed from any one or more of the
aforementioned particular peptides.
Favourably, the invention includes a peptide formed from any of two
contiguous m~mber.~ of the aforementioned particular peptides.
As stated, the invention also extends to the functional derivatives, analogues
and variants of the peptides mentioned herein:
Functional derivatives includes salts and solvates of the peptides mentioned
herein and also the peptides of the invention chemically modified by the ~tt~chmenf of
groups or moieties so as to improve the physical properties, such as stability, or the
therapeutic properties, for example the ph~rrn~-~okinetic ~lo~ lies, of the protein.
Functional analogues includes functionally analogous peptides wherein one or
more amino acids of the peptides mentioned herein are replaced with alternative
amino acids.
~it~rn~tive amino acids includes amino acids of alternative stereochemistry to
the amino acids in ob protein.
Functional analogues also include small molecule agonists or antagonists of
the peptides mentioned herein. Such compounds may be prepared and tested
according to known procedures, for example those disclosed in GB2292382.
Salts include ph~rm~-elltically acceptable salts, especially ph~rrn~rentically
acceptable acid addition salts.
Acid addition salts of the peptides are p~ d in a standard manner in a
suitable solvent from the parent compound and an excess of an acid, such as
hydrochloric, hydrobromic, sulphuric, phosphoric, acetic, maleic, succinic, or
meth~n~snlrhonic. The acetate salt form is especially useful. Certain of the
compounds form inner salts or zwitterions which may be acceptable. Cationic salts
are prepared by treating the parent compound with an excess of an ~k~iine reagent,
such as a hydroxide, carbonate or ~Ik~xide conf~inin~ the ~ opliate cation. Cations
such as Na+, K+, Ca2+ and NH4+ are examples of cations present in
ph~rm~l eutically acceptable salts.
Solvates include pharrnaceutically acceptable solvates, such as lly~lldl~s.
It will be appreciated that the invention includes both peptide and non-peptide
compounds.
In addition the invention includes sub-fr~ment~ of the particular peptides
ob21-26,ob27-32,ob33-36,ob37-41,ob42-54,obSS-56,obS7-74,ob93 -105,
oblO6 -115, obll6 -149 and oblS~-167, especially obS7-74;ora peptide forrned
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W O 97/46585 PCT~P97/02968
from any one or more, especially of any two contiguous members, of the said
particular peptides; or a functional derivative, analogue or variant thereof.
Suitable peptides or sub-fragments comprise at least 4 amino acids.
The peptides of the invention are suitably prepared by using conventional
t 5 digestion methods, synthetic techniques or by use of standard t;~rc;ssion
methodology.
Thus in a further aspect, the present invention provides a process for the
epald~ion of a peptide, or a functional derivative thereof, the process comprising the
steps of:
hydrolysing the peptide, especially an ob protein and in particular a hurnan ob protein,
into at least two peptide fr~mentc;
sep~ g the peptide fr~gment.~; and optionally thereafter y~ g a functional
derivative thereof.
The hydrolysis of the protein is suitably effected by enzymic digestion, using
for exarnple trypsin.
The separation of the required peptide is conveniently accomplished by use of
an a~lv~liate chromatographic means, such as column chromatography.
The specific reaction conditions for the tre~tment of the ob protein, providing
they are commensurate with the stability of the required product, are determined by
the nature of the particular reagent used, for exarnples when trypsin is the reagent then
the reaction is normally carried out within a temperature range of 25~0~C and a pH
range of 7-9, preferably at 37~C and pH 7.4.
As stated, the peptides of the present invention may also be prepared by
conventional synthetic procedures, for example by use of liquid or solid-phase peptide
synthesis.
Accordingly, the present invention provides a synthetic peptide or a functional
derivative, analogue or variant thereof, which modulates body weight, substantially be
means of mocl~ ting energy lltili~tion.
Any of the peptides mentioned herein form part of the inventiorl as synthetic
peptides.
Peptide bonded units of the proteins associated with the present invention can
be prepared by standard peptide synthesis techniques using a peptide synth~ r
(Atherton, E. and Sheppard, R.C. (eds.) (1989) Solid Phase Peptide Synthesis: A
practical approach, LRL Press, Oxford) followed by procedures a~plu~liate to direct
disulphide or amide bond formation.
Methods of well-known peptide synthesis are set forth by Ali et. al., J. ~.
~h~m., ~:984 (1986) and J. ~çs~. Chem., 30:2291 (1987) and are incorporated by
reference herein. Preferably, the peptides are ;~re~ ,d by the solid phase technique ûf
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WO 97/46585 PCT/3EP97/02968
Merrifield (I. Am. ~ ., 8$:2149 (1964)3. However, a combination of solid
phase and solution synthesis may be used, as in a convergent synthesis in which di-,
tri, tetra-, or penta-peptide fr~gm/ nt~ may be prepared by solid phase synthesis and
either coupled or further modified by solution synthesis.
S During synthesis, the side chain functional groups (e.g., -NH2, -COOH, -OH,
-SH) are protected during the coupling reactions. Normally, the .~-amino group is
temporarily protected as fluorenylmethoxycarbonyl (Fmoc) but other acid- or base-
labile protecting groups can be used, e.g., t-Butoxycarbonyl (Boc). The amino side
chain group of lysine is protected as t-buL?~yc~L?onyL benzyloxycarbonyl or
p-chlorobenzyloxycarbonyl (Z or Cl-Z). Acetarnidomethyl, trityl, t-butyl, S-t-butyl or
para-methylbenzyl (p-MBz) protection is used for cysteines. Hydroxy groups are
protected as butyl or benzyl ethers and carboxyl groups are protected as butyl, benzyl
(Bz) or cyclohexyl esters.
The peptides can be synthesi7~-n either from the C-t~r r.inll~ or the
N-terminus, preferably the former. Prior to coupling the alpha-carboxyl group (of a
suitable protected arnino acid~ is activated. One skilled in the art can activate the
protected group in a number of ways. For example, one may use N,N'
dicyclohexylcarbodiimide (DCC), 2(1 H-benzotriazol- 1 -yl)- 1,1,3,3 -tetramethyluronium hexafluorophosphate (HBTU), p-nitrophenyl esters ~pNp),
hydroxybenzotri~ole ester (HOBt), N-hydroxy succinimidyl ester (OSu) mixed
anhydride or symmetrical anhydride.
Solution synthesis of peptides is accomplished using conventional methods
to form amide bonds. Typically, a protected Boc-amino acid which has a free
carboxyl group is coupled to a protected amino acid which has a free amino groupusing a suitable carbodiimide coupling agent, such as N,N' dicyclohexyl carbodiimide
(DCC), optionally in the presence of l-hy~ o~yberlzotriazole (~OBT) and
dimethylamino pyridine (DMAP~.
In solution phase synthesis, the coupling reactions are preferably carried out
at low temperature (e.g., -20~C) in such solvents as dichloromethane (DCM),
dimethyl formamide (DMF), N-methyl pyrrolidone (NMP), tetrahydrofuran (T~IF)
acetonitrile (ACN) or dioxane.
If solid phase methods are used, the peptide is built up sequentially starting
from the carboxy 1... ~ and working towards the amino ~ c of the peptide.
Solid phase synthesis begins by covalently ~S3chin~ the C terminlls of a protected
35 amino acid to a suitable resin, such as 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxy resin (Rink amide resin, H.Rink, Tetrahedron Letters 28,3787, (1987)), 4-
benzyloxybenzyl alcohol resin (Wang resin, S.S. Wang, JACS, 9$, 1328, (1973)) or 4-
hydroxymethyl phenoxy acetic acid resin.
CA 022~7240 1998-12-04
W 097146585 PCT~EP97/02968
In the solid phase synthesis, the first arnino acid residue is normally attachedto an insoluble polymer. For exarnple, two commonly used polymers are po}ystyrene
(1% cross-linked with divinyl benzene) and 1% cross-linked polyacrylamide. Thesepolymers are function~ ed to contain a reactive group, e.g., -OH, -NH2 and -C~2CI
5 to link the first arnino acid of the targeted peptide (i.e., carboxy terminus). The choice
of the linkage between the first arnino acid and the polymer is dictated by the carboxy
terminns of the peptide. For example, peptides having a carboxyl group at the
C-t~rmin-l~ would be linked by an ester linkage and for peptides with a carboxamide
ending would have an amide linkage.
Once the first protected amino acid has been coupled to the desired resin, the
a arnino protecting group is removed by tr~tment with a secondary amine such as
piperidine, and the free carboxyl of the next (protected) amino acid is coupled to this
amino group. This process is carried out sequentially, without isolation of the
intermediate, until the peptide of interest has been formed. The completed peptide
may then be deblocked and/or cleaved from the resin in any order.
Preferred solvents for the coupling reactions include, but are not limited to,
dichlororneth~ne (DCM), dimethyl formamide (DMF) and N-methyl pyrrolidone
(NMP). After the desired sequence is synth~ e~l the peptide is deprotected and
cleaved from the resin using trifluoroacetic acid or trifluoromethane sulphonic acid.
The preferred method for cleaving a peptide from the support resin is to treat
the resin supported peptide with trifluoroacetic acid in the presence of suitable cation
and carbonium ion scavengers such as phenol, anisole, thioanisole, ethane dithiol,
water or ethylmethyl sulphide.
To obtain the compounds of the present invention, the synthetic peptides may
be cyclized/coupled using methods well known in the art.
For example coupling via a disulphide bond of two linear peptides both
co~-t~ g cysteine residues may be achieved in a selective manner by reaction of the
free thiol on one chain with a suitably activated cysteine derivative on the other chain.
A group which is especially useful as a displaceable protecting group is the S-
(carbomethoxy- sulphenyl) derivative. Examplary of this method is the protection of
both linear peptides' cysteine residues with the acetamidomethyl (Acm) group.
Tre~tment of one chain with mercury (II) acetate followed by beta mercaptoethanol
removes the ~ et~midomethyl protecting group. Tre~tment of the second chain withcarbomethoxysnlrhenyl chloride gives the activated species. Stirring of the two
J 35 peptides in dilute aqueous solution at a pH of about 7 to 8 causes displacement of the
carbomethoxysulphenyl group and formation of the interchain disnlphi~le
If an intramolecular ~ llphicle is to be formed then the col~e~ponding linear
peptide can be completely de~lott;.i~ed and produced as a dim~_rca~ . Any oxidizing
CA 022~7240 1998-12-04
W 097/4658~ PCT~P97/02968
agent known in the art to be capable of converting a dirn~lca~Lall to a disulphide may
then be used. Examplary of such agents are an alkali metal ferricyanide, (e.g.,
potassium or sodium ferricyanide), oxygen gas, diiodomethane or iodine. The
reaction is conclllcted in a suitable inert solvent, such as a(lueous methanol or water, at
teln~ Lu~s from about 0 to 40~C, under high dilution. The pH is usually m~int~ined
at about 7 to 8. Cyclisation may be perforrned upon the peptide while it is still
s~ttsu.h~ to the support resin or while other functional groups are still protected, but it
is preferably performed on the deprotected free peptide.
In cases where two ~ ulrhi~l~s are to be formed between two linear peptides,
two types of cysteine thiol protecting groups can be employed eg Acm and trityl.Each peptide would contain one of each type arranged so that one pair of cysteines to
be coupled are protected with kityl groups and the other pair with Acm. Independent
removal of the trityl group from each peptide would give two sepa,~L~ monothiol
derivatives which can be coupled by activating the thiol on one peptide with
2~2~dipyridyl~lie~llrhi~le and then adding the other monothiol peptide to give the bis(S-
~cet~mido- methyl)disulphide-linked peptide. The second disulphide can be obtained
by direct iodine oxidation of this product as described by Kamber (B. Kamber, Helv.
Chim. ~cta 54, 927, (1971)), and Kamber et. al. (B. Kamber et. al., Helv. Chim. Acta
~i3, 899, (1980)).
Peptide chains can also be coupled using a linking group such as -
NH(CH2)nCO-. This is most easily achieved by employing the Na-Fmoc derivative
of the corresponding amino acid (NH2(CH2)nCOOH) and incorporating it into the
growing peptide chain during conventional solid phase synthesis. A similar strategy
can be employed to couple peptide chains using the side chain carboxyl Oran acidic
amino acid such as glutamic acid, and the side chain amino of a basic amino acid such
as lysine. In this case compounds such as the N6-g glutamyllysine derivative below
may be incorporated into the growing peptide chain during conventional solid phase
synthesis
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CH3CON ~-CH-CO OH
I
(CH2)2
I
S CO
,,
NH
(CH2)4
FmocNHCHCONH2
Coupling to the growing peptide chain is through the a carboxyl of the glutamic acid
residue and removal of the Fmoc grouping on the lysine a amino group provides a
starting point for addition of further amino acids.
Altern~tively the Na-trityl protecting group may be employed on the
glutamic acid residue and after coupling this may be removed with 80% acetic acid
and N-acetylated with acetic anhydride. Further couplings may proceed as previously
described.
N-tf nnin~l N-acetyl groups may be introduced by acetylation of the free
amino proteinated by removal of the amino protecting group, with acetic anhydride.
C-termin~l carboxamide groups are obtained by using an ~l,lo~liate solid phase
synthesis resin such as the Rink amide resin.
As stated the peptides of the invention may also be prepared using
recombinant DNA techniques by expression of DNA encoding the polypeptide
sequence.
Accordingly, the invention extends to a recombinant peptide or a functional
derivative, analogue or variant thereof, which modulates body weight, substantially be
means of mo~ t;n~ energy utilisation.
Any of the peptides mentioned herein form part of the invention as
recombinant peptides.
In a further aspect, the invention provides a process for pl~ep~illg a
compound according to the invention which process compri~es t;~l.,s~ g DNA
encoding said compound in a recombinant host cell and recovering the product.
The DNA polymer comprising a nucleotide sequence that encodes the
compound also forms part of the invention.
The process of the invention may be performed by conventional recombinant
techniques such as described in Maniatis et. al., Molecular Cloning - A Laboratory
CA 02257240 1998-12-04
W O97t4658~ PCTAEP97/02968
Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, II and III (D.M. Glovered., IRL Press Ltd).
In particular, the process may comprise the steps of:
i) preparing a replicable expression vector capable, in a host cell, of expressing
S a DNA polymer comprising a nucleotide sequence that encodes said compound,
ii) transforming a host cell with said vector;
iii) cllltl~ring said transformed host cell under conditions permitting ~Jression of
said DNA polymer to produce said compound; and
iv) recovering said compound.
The invention also provides a process for ~ ali~lg the DNA polymer by the
con(len~tion of ~plol.liate mono-, di- or oligomeric nucleotide units.
The pr~dl~lion may be carried out chemically, enzymatically, or by a
combination of the two methods, in vitrQ or in vivo as a~ liate. Thus, the DNA
polymer may be prepared by the enzymatic of aL~ropliate DNA frS~ment~, by
conventional methods such as those described by D. M. Roberts et ~ in Bioc hemi~try
1985, 24, 5090-5098.
The DNA fragments may be obtained by digestion of DNA cont~ining the
required sequences of nucleotides with a~iopliate restriction enzymes, by chemical
synthesis, by enzymatic polymerisation on DNA or RNA templates, or by a
combination of these methods. Preferably total synthesis of DNA fr~gment~ would be
employed.
Digestion with restriction enzymes may be l,c~ led in an a~ Jpliate
buffer at a temperature of 20~-70~C, preferably in a volume of SOml or less with0.1-1 Omg DNA.
Enzymatic polymerisation of DNA may be carried out in vitro using a DNA
polymerase such as DNA polymerase I (Klenow fragment) in an a~pr~pliate buffer
co~ g the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at
a telllp~l~LI~le of 10~-37~C, proteinrally in a volume of SOml or less.
Enzymatic ligation of DNA fr~gm~nt~ may be carried out using a DNA ligase
such as T4 DNA ligase in an a~ .liate buffer at a temperature of 4~C to ambient, in
a volume of 50ml or less.
The chemical synthesis of the DNA polymer or fnq~ment~ may be carried out
by collv~llLional phosphotriester, phosphite or phosphoramidite rhemi~tly, using solid
phase techniques such as those described in 'Chemical and Enzymatic Synthesis ofProtein Fr~gn~entc - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag
Chemie, Weinheim (1982),or in other scientific publications, for exarnple M.J. Gait,
H.W.D. Matthes, M. Singh, B.S. Sproat, and RC. Titmas, Nucleic Acids Research,
1982, 10, 6243; B.S. Sproat and W. B~lllw~lh, Tetrahedron Letters, 1983, ~L, 5771;
CA 022~7240 1998-12-04
W 097/46585 PCT~EP97/02968
M.D. Matteucci and M.H Caruthers, Tekahedron Letters, 1980, ~, 719; M.D.
Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981, 103,
3185; S.P. Adams ç~ ~Ll., Journal of the American Chemical Society,1983, 105, 661;
N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984,
S L~, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably an
automated DNA synthe~i7er is employed.
The DNA polymer is preferably prepared by lig~tin~ two or more DNA
molecules which together comprise a DNA sequence encoding the compound.
The DNA molecules may be obtained by the digestion with suitable
restriction enzymes of vectors carrying the required coding sequences.
The precise skucture of the DNA molecules and the way in which they are
obtained depends upon the skucture of the desired product. The design of a suitable
strategy for the construction of the DNA molecule coding for the compound is a
routine matter for the skilled worker in the art.
The expression of the DNA polymer encoding the compound in a
recombinant host cell may be carried out by means of a replicable expression vector
capable, in the host cell, of expressing the DNA polymer. The ~ c~ion vector is
novel and also forms part of the invention.
The replicable e~ples~ion vector may be prepared in accordance with the
invention, by cleaving a vector compatible with the host cell to provide a linear DNA
segment having an intact replicon, and combining said linear segment with one ormore DNA molecules which, together with said linear segment, encode the
compound, under ligating conditions.
The ligation of the linear segment and more than one DNA molecule may be
carried out simultaneously or sequentially as desired.
Thus, the DNA polymer may be preformed or formed during the conskuction
of the vector, as desired.
The choice of vector will be determined in part by the host cell, which may
be prokaryotic, such as 1~. ~11, or eukaryotic, such as mouse C127, mo'use myeloma,
chinese h~m~tPr ovary, fungi e.g. fil~ment-~us fungi or unicellular yeast or an insect
cell such as Drosophila. The host cell may also be in a transgenic animal. Suitable
vectors include pl~mic~, bacteriophages, cosmids and recombinant viruses derivedfrom, for example, baculoviruses or vaccinia.
The pl~a d~ion of the replicable expression vector may be carried out
conventionally with a~uropliate enzymes for restriction, polymeri~tion and ligation
of the DNA, by procedures described in, for exarnple, Maniatis ~ al., cited above.
Polymerisation and ligation may be performed as described above for the p~ )a.~ion
of the DNA polymer. Digestion with restriction enzymes may be performed in an
CA 022~7240 1998-12-04
W 097/46585 PCTrEP97/02968
ol,liate buffer at a temperature of 20~-70~~, proteinrally in a volume of 50ml or
less with 0.1-lOmg DNA.
The recombinant host cell is prepared, in accordance with the invention, by
transforming a host cell with a replicable e~ s~ion vector of the invention under
5 transforming conditions. Suitable kansforming conditions are conventional and are
described in, for example, Maniatis et al., cited above, or "DNA Cloning" Vol. II,
D.M. Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions is deterrnined by the host cell. Thus, a
kact~-.ri~l host such as F" coli may be treated with a solution o~CaC12 (Cohen ~ ~Ll,
Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of
RbCl, MnC12, potassium acetate and glycerol, and then with 3-[N-morpholino]-
propane-sulphonic acid, RbCl and glycerol. 1~51mms~ n cells in culture may be
transformed by calcium co-precipitation of the vector DNA onto the cells.
The invention also extends to a vector comprising a compound of the
invention.
The invention also extends to a host cell transformed with a replicable
.es~ion vector of the invention.
Culturing the transformed host cell under conditions permi~tin~ ,s~ion of
the DNA polymer is carried out conventionally, as described in, for example, Maniatis
et ~ and "DNA Cloning" cited above. Thus, preferably the cell is supplied with
nutrient and cultured at a te~peldlLlle below 45~C.
The expression product is recovered by conventional methods according to
the host cell. Thus, where the host cell is bacterial, such as E. ~521i it may be lysed
physically, chemically or enzymatically and the protein product isolated from the
resulting lysate. If the product is to be secreted from the bacterial cell it may be
recovered from the periplasmic space or the nutrient medium. Where the host cell is
m~mm~ n, the product may proteinrally be isolated from the nutrient medium.
The DNA polymer may be assembled into vectors designed for isolation of
stable transforrned m~n~m~ n cell lines c;~le3~ g the product; e.g. bovine
papillomavirus vectors or amplified vectors in chinese harnster ovary cells (DNAcloning Vol.II D.M. Glover ed. IRL Press 1985; ~nfmRn, R.J. ~ ~1., Molecular andCellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Procee~lings of
the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D.V. et ~.,
European Patent Application No. 0093619, 1983).
The peptides prepared by use of the above mentioned methods can, as
required, be purified by a number of techniques. Preferred embodiments include gel
filtration, chromatogaphy, reverse phase HPLC and cryst~ tion, especially
chromatogaphy is used. The purified products can then be analysed for purity using
-10-
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W O 97/46585 PCTAEP97/~2968
HPLC, arnino acid analysis, amino acid sequencing and fast atom bombardment
and/or electrospray mass spectrometry.
The functional derivatives, analogues and variants of the proteins mentioned
herein may be prepared by using conventional methods analogous to those mentioned
5 herein.
As stated, the compounds of the invention are indicated to have useful
ph:~rm~ .eutical plo~-e~lies. Accordingly, there is also provided a compound of the
invention for use as an active therapeutic substance.
In particular the compounds of the invention are considered to be capable of
10 moclnl~ting body weight substantially by means of enhancing energy utilization and
are therefore of potential use in the trçatmenl of nutritional and metabolic disorders,
particularly obesity and diabetes.
The invention also provides a method for the treatment of nutritional and
metabolic disorders, which method comprises the ~(imini~tration of an effective,15 ph~rrnz~eutically acceptable and non-toxic amount of a compound of the invention.
The invention therefore further provides a ph~rmsl~eutical composition
comprising a compound of the invention and a phs~rm~/~.elitically acceptable carrier.
In use the active compound will normally be employed in the form of a
ph~rm~ceutical composition in association with a human or veterinary pharmaceutical
20 carrier, diluent and/or excipient, although the exact form of the composition will
depend on the mode of a~1mini~tration. The active compound may, for example, be
employed in the forrn of tablets, capsules, lo~:enges or syrups for oral ~imini~tration;
in the form of snuff, aerosol or nebulisable solution for inhalation; in the form of
sterile~solutions for parenteral a~lmini~tration, or in the form of creams, lotions,
25 liniments, gels, ointments or sprays for topical ~tlmini.ctration. P~ent~,dl routes of
a~lmini~tration include inkavenous, intramuscular, subcutaneous, transcutaneous and
intraperitoneal arlmini~tration.
Also included are formulations of the above derivatives suitable for use in
subcutaneously implanted pumps or controlled release devices, in transdermal patches
30 and as micronised powders suitable for intranasal a-1mini~tration.
The dosage ranges for a~1mini.~tration of the compounds of the present
invention are those to produce the desired effect on the condition to be treated, the
dosage will proteinrally vary with age, extent or severity of the medical condition and
contraindications, if any. The dosage can vary from O.OOlmg/kg/day to SOmg/kg/day,
35 but preferably 0.01 to I .Omg/kg/day.
~ ;olid oral dosage forms may contain conventional excipients such as
diluents, for example lactose, microcrystalline cellulose, dicalcium phosphate,
mannitol, m~gne~ium carbonate, glycine, dextrose, sucrose, starch, m~nnitnl, sorbitol
CA 022~7240 1998-12-04
W097/46S85 PCT~P97/02968
and calcium carbonate; binders, fo} example liquid glucose, syrup, acacia, gelatin,
starch mt1c.il~ge, methylcellulose, polyvinylpyrrolidone, ~Igin~tes, and pregel~tini~ecl
starch, cl;~in~egrants for example starch, alginic acid, microcrystalline cellulose,
pectin, cross-linked polyvinylpyrrolidone, sodium starch glycollate and sodiurn
carboxymethyl-cellulose; glidants for example talc and silica; lubricants for example t
stearic acid and magnesium stearate; pl~s~ es for example sorbic acid and methylor propyl parahydroxyben~c ~fe, or ph~rm~eutically acceptable wetting agents forexample sodium lauryl sulphate.
Capsules consist of a shell, normally of gelatin together with other
ingredients for example, glycerol, sorbitol, surface-active agents, opaque fillers,
preservatives, sweeteners, flavours and colours. The contents of capsules may include
diluents, lubricants and disintegrants. Tablets consist of compressed powders orgranules, may be coated or uncoated and may be ~1eci~n~c~ so as to dissolve, disperse
or effervesce before ~-lmini~tration to the patient, or to dissolve or disperse in the
gastrointestinal tract either immediately after swallowing, or, for example in the case
of tablets with acid-insoluble co~tings~ at later times. Tablets usually containexcipients such as diluents, binders, disintegrants, glid~nt~, lubricants and may
contain colours and flavours. Effervescent tablets proteinrally contain acids together
with carbonates or bicarbonates. Coatings for tablets may consist of natural or
synthetic resins, gums, insoluble fillers, sugars, plasticisers, polyhydric alcohols and
waxes and may also contain colours and flavours. Lozenges and pastilles are intended
to dissolve in the mouth. Lozenges may be moulded or compressed, and usually have
a flavoured base. Pastilles are moulded from a base of gelatin and glycerol or acacia
and sucrose. They may contain a preservative as well as colours and flavours.
Film-coating resins include cellulose derivatives, :zein, vinyl polymers and
acrylic resins, and coating compositions usually include plasticisers, such as castor oil
or glycerol triacetate. Enteric-coating resins include cellulose acetate phth~l~te and
copolymers of methacrylic acid.
Solid compositions suitable for oral a~1mini~tration may be obtained by
conventional methods of blending, filling, granulation, tabletting or the lilce.~epeated blending operations may be used to distribute the active agent throughout
those compositions employing large qu~ntities of fillers.
Liquid compositions suitable for oral ~-lmini~tration may be in the form of,
for example, elixirs, mixtures, concentrated solutions, suspensions, emulsions or
linctl~es They may be presented as a dry product for reconstitution with water or
other suitable vehicle before use. Such liquid compositions may contain conventional
excipients such as suspending agents, for exarnple sucrose, sorbitol, gelatin, methyl
cellulose, carboxymethylcellulose, hydroxypropyl methyl cellulose, sodium ~Igin~tP7
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CA 022~7240 1998-12-04
W 097/~658~ PCTrEP97/02968
Xanthan gum, acacia, carageenan, silica, aluminium stearate gel; emulsifying agents,
for example lecithin, acacia, sorbitan mono-oleate; aqueous or non-aqueous vehicles
which include edible oils, oily esters, for example esters of glycerol, ethanol, glycerol;
burr~ lg agents for exarnple citrates and phosphates of alkali metals; preservatives,
for example sodium benzoate, sorbic acid, methyl or propyl parahydroxybenzoate;
and if desired, conventional flavouring and colouring agents.
The composition may be implanted subcutaneously, for example in the forln
of a coll.~lGssed tablet or slow release capsule.
Alternatively, compositions suitable for injection may be in the form of
solutions, suspensions or emulsions, or dry powders which are dissolved or suspended
in a suitable vehicle prior to use.
Fluid unit dosage forrns are prepared ut;lising the compound and a
pyrogen-free sterile vehicle. ~he compound, depending on the vehicle and
concentration used. can be either dissolved or suspended in the vehicle. Solutions
may be used for all forrns of parenteral ~tlmini~tration, and are particularly used for
intravenous infection. In preparing solutions the compound can be dissolved in the
vehicle, the solution being made isotonic if necessary by addition of sodium chloride
and sterilised by filtration through a sterile filter using aseptic techniques before
filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution
stability is adequate, the solution in its sealed containers may be sterilised by
autoclaving. Advantageously additives such as buffering, solubilising, stabilising,
preservative or bactericidal, suspending or emulsifying agents and/or local anaesthetic
agents may be dissolved in the vehicle.
Dry powder~ which are dissolved or suspended in a suitable vehicle prior to
use may be preparcd by filling pre-st~rili~e~l drug substance and other ingredients into
a sterile container using aseptic technique in a sterile area. Alternatively the drug and
other ingredients may bc dissolved in an aqueous vehicle, the solution is sterilised by
filtration and distributcd into suitable containers using aseptic technique in a sterile
area. The product is then freeze dried and the containers are sealed aseptically.
Parenteral suspensions, suitable for intramuscular, subcutaneous or
intradermal injection, are pl~aled in sllhstzmti~lly the sarne manner, except that the
sterile compound is suspended in the sterile vehicle, instead of being dissolved and
st~rili~tion cannot be accomplished by filtration. The compound may be isolated in a
sterile state or ~ltf~rn~tively it may be sterilised after isolation, e.g. by gamma
irradiation. Advantageously, a suspending agent for example polyvinylpyrrolidone is
included in the composition to facilitate uniform distribution of the compound.
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CA 02257240 l998-l2-04
W 097/46585 PCT~EP97/02968
In a further aspect there is provided a method of treating nukitional and
metabolic disorders, which comprises ~lmini.~tt?ring to the ~urr~ ~ an effective, non-
toxic amount of a compound of the invention.
The invention also provides the use of a compound of the invention for the
5 m~nllf~ctll~e of a medicament for keating nutritional and metabolic disorders, such as
obesity and diabetes.
No unexpected toxicological effects are expected when compounds of the
invention are ~-1mini.clered in accordance with the present invention.
The following examples illustrate compounds of the invention.
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W 097/4658S PCT~EP97tO2968
Pharmacological Methods: The activity of the compounds of the invention are
assessed according to the methodology set our be~ow:
5 I~FFECT OF I.EPTIN FE~AGMI~NTS ON FOOD INTAKE IN SD RATS
Surgely
Rats are pre-treated with Synulox (O. lml/lOOg) approx l hour before anaesthesia, and
then ~n~esthetised with Domitor (0.04ml/lOOg i.m.) and sublimase (0.9ml/lOOg i.p.)
10 Each rat has a c~nn~ implanted stereotaxically into the lateral brain ventricle under
sterile conditions. ~n~esthesi~ is then reversed using Antisedan and Nubain (50% v/v
: 50% v/v 0.02ml/l OOg) I.P. After surgery each rat receives 0.05 ml Zenecarp.
Experimental procedure:
Experiment l: Following surgery the body weight of each animal was monitored
daily throughout the procedure.
In order to verify that the cannula was in the lateral ventricle, Angiotensin II(lOOng/5~u1) was injected icv and water intake was monitored for 5 min after
20 injection.
24 hour food intake was measured on day 5 and 6 after surgery. On day 6 the ~nim~l.s
were divided according to their body weight into 3 groups (a,b and c, 8 rats per group)
and then fasted overnight.. On the day of e~ ent (day 7) rats were injected icv as
follows::
25 group a-vehicle (PBS, phosphate buffer solution, S,ul /rat);
group b- human leptin (l l.5 ~Lg/5~1); and
group c- leptin tryptic digest (30,ug/5~
A known quantity of food in excess of the daily requirement was supplied to the rats
irnmediately after the icv injection procedure was completed.. Food intake and body
30 weight were then measured 24h later.. The results obtained are shown in Table l .
Experiment 2: A separate group of animals was prepared exactly as described above
but on the day of experiment after overnight fast, groups of rats were injected as
follows:
35 group a-vehicle (PBS 5,ul /rat);
group b-human leptin (8.75 ,ug/5,ul);
group c-murine leptin ( l O ,ug/5~
group d-ob 57 -74, sequence VTGLDFIPGLHPILTLSK (3.33,ug/5~1);
Again a pre-weight quantity of food, in excess of the daily requirement, was re-given
40 following the injection; change in body weight and the quantity of food consumed
were recorded 24h later. The results obtained are shown in Table 2.
CA 02257240 1998-12-04
W 097/46585 PCT~EP97/02968
Results:
Table 1. Ef~ect of human leptin and its fragments given
intracerebroventricularly on body weight and food intal~e
5 in SD rats. * P<0.05.
24h food intake Bwt Change
(g) (g/24h)
Vehicle (5~1/rat, n=8) 36.68 +2.5 31.44 + 1.2
h-Leptin 30.62~1.5* 22.88 i 2.05*
(11.5 ~lg/rat, n=8)
h-Leptintryptic 34.6 i: 1.99 23.88 + 1.2*
digest
(30 ~Lg/rat, n=8)
10 Table 2. Effect of human leptin (h-leptin), murine leptin (m-leptin) and leptin
fragment 57-74 (VTGLD~IPGLHPILTLSK) given intracerebroventricularly on body
weight and food intake in SD rats. * P<0.05.
24h food intake Bwt Change
(g) (g/24h)
Vehicle (5~Ll/rat, n=8) 32.16 +0.8 30.55 ~t 0.67
h-Leptin 33.7+1.6 24.5 i 3.7
(8.75 ~Lg/rat, n=8)
m-Leptin 31.3 :t 1.7 23.7 + 2.18
(10 ~g/rat, n=8)
Leptin fragment 33.6 + 0.9 26.8 + 1.2*
57-74 (3.33 ,ug/rat,
n=8)
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