Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF THE BLOOD-BRAIN BARRIER
TRANSPORTER FOR LEPTIN
RELATED APPLICATIONS
This application claims priority from U.S. Provisional Patent
Application Serial No. 60/150,300, filed August 23, 1999.
FIELD OF THE INVENTION
The present invention relates generally to modulating the body weight
and/or appetite of mammals, including humans. More particularly this invention
relates to compositions and methods which modulate the transport of weight-
controlling molecules, such as leptin, across the blood-brain barrier.
GOVERNMENT RIGHTS
This invention was made with support from the United States
Government. The United States Government retains certain rights to this
invention.
BACKGROUND OF THE INVENTION
Obesity is defined as an excess of body fat relative to lean body mass
and is associated with important psychological and medical morbidities,
including
hypertension, elevated blood lipids, and diabetes. Body weight and energy
balance
are thought to be regulated by a feedback mechanism in which the regions of
the
brain, for example, the hypothalamus, senses the amount of energy stored in
the body
then adjusts food intake and activity level accordingly [Brobeck, J.R., Yale
J. Biol.
Med, 20:545-552 ( 1948)]. Early experiments showed that the arterial transfer
of
blood from one animal having a hypothalamic lesion to a normal healthy animal
resulted in the reduction of food intake by the normal animal [Hervey, G. H.,
.l.
Physiol., 145:336-352 (1959)]. From these results it was hypothesized that at
least
one component of the feedback mechanism circulated through the bloodstream and
that the component acted on the brain. It has been suggested that the OB gene
may be
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responsible for the generation of this blood-borne factor [see also Coleman,
D.L.,
Diabetologica, 14:141-148 (1978)].
Recent studies of the OB gene have confirmed that the OB gene
product known as leptin is the blood-borne factor which works to maintain body
weight and energy balance [Zhang et al., Nature., 372:425-432 (1994); and
Freidman
et al., PCT Application No. PCT/US95/10479]. Further, it has also been shown
that
the administration of leptin results in a decreased amount of body fat
[Pelleymounter,
M.A. et al. Science, 269:540-543 (1995); Halaas, et al. Science, 269:543-546
(1995);
Campfield, et al. Science, 269:546-549 (1995)]. It is believed that leptin
acts on the
brain to inhibit food intake, regulate energy expenditure, and control body
weight.
In order for leptin to play this type of role, leptin must cross over the
blood-brain barrier to enter the brain. The amount of leptin sensed by the
brain results
from a combination of the permeability of the blood brain barrier and the
amount of
leptin in the bloodstream which in turn depends on the level of stored energy
or body
fat of an individual [Considine, R. V. et al., N. Eng. J. Med. 334:292-295
(1986)].
Obesity can occur when the brain incorrectly senses a low level of leptin and
so
initiates mechanisms to raise that level by increasing the amount of body fat.
This
cycle usually continues until the brain senses an appropriate amount of leptin
at which
time the body weight ceases to increase. As described herein, it is believed
that
increasing the efficiency of leptin transport across the blood-brain barner
would be an
effective treatment for obesity, in most cases.
Blood-borne leptin is able to enter the brain because of the presence of
a specific saturable transporter located at the blood-brain barrier [Banks et
al.,
Peptides 17(2):305-311 (1996)]. Because leptin is a large protein, leptin in
the blood
would be largely excluded from the brain in the absence of such a transporter.
It is
believed that the transporter is close to or contains within its structure
some sites
which, when activated, modify the transport rate of leptin. Such sites,
conceptually
analogous to cofactors binding sites for enzymes or allosteric regulatory
sites for
receptors, provide therapeutic targets which can be manipulated to alter the
rate of
leptin transport from the blood into the brain so as to control body weight.
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The mechanism of transport of proteins and peptides such as leptin
across the blood-brain barrier is poorly understood. Some proteinsipeptides
cross this
barrier by diffusing directly through the endothelial/ependymal membranes
according
to their lipophilicity and/or molecular weight with smaller lipophilic
molecules
passing more freely [Banks & Kastin, Psvchoneuroendocrinology, 10:385-399
(1985)]. Others are transported by saturable, carrier-mediated systems [Banks
&
Kastin, Pharmacol. Biochem. Behav., 21:943-946 ( 1984)], such as the one that
transports Tyr-MIF (Tyr-Pro-Leu-Gly-amide) [Banks & Kastin, J. Pharmacol. Exp.
Ther, 239:668-672 ( 1986)]. Another mechanism by which proteins or
polypeptides
cross the blood-brain barrier is through receptor-mediated permeabilization
which is
enabled by the administration of molecules such as bradykinin, leukotrienes,
histamine, and 5-hydroxytryptamine [Unterberg, A., J. Cereb. Blood Flow
Metab.,
4:574-585 (1984)]. Similarly, molecules such as leucine encephalin, a-
adrenergics,
arachidonic acid, aluminum, phorbomyristate esters, and a-thrombin increase
blood-
brain barrier permeability while angiotensin II and ~3-adrenergics reduce the
permeability [Grieg, N., Physiology and Pharmacology of the Blood-Brain
Barrier
Handbook of Experimental Pharmacology, 103:487-523 Springer-Verlag, Berlin
(1992)].
There is a considerable need for molecules or compositions and
methods for using those molecules and compositions which modulate (i.e.,
enhance or
inhibit) the transport of weight-controlling molecules, such as leptin, across
the blood-
brain barner.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for
modulating feeding behavior and/or appetite in mammals as well as for
modulating
body weight in mammals. More particularly, the present invention is directed
to the
methods and compositions for modulating (enhancing or inhibiting) the
transport of
leptin across the blood-brain barrier and across other blood/tissue barriers.
The
invention is also directed to methods and compositions for modulating
(increasing or
decreasing) body weight and/or metabolism by altering the transport of leptin
across
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the blood-brain barrier. According to the invention, leptin transport across
the blood-
brain barrier may be increased, thereby resulting in a reduction in body
weight, and/or
a decrease in appetite. Conversely, leptin transport across the blood-brain
barrier may
be decreased (or inhibited) resulting in an increase in appetite and/or body
weight in
patients in need thereof (e.g., anorexia, cachexia of aging, tumor-induced
cachexia).
The compositions may act on either side of the blood-brain barner (or other
blood-
tissue barriers) to result in altered transport of leptin, although preferred
compositions
and methods act on the blood side of the barner.
A preferred method of the invention comprises administering to a
subject in need thereof a composition which comprises an adrenergic agonist in
an
amount effective to increase the transport of leptin or leptin variants,
analogs,
fragments, consensus leptin, or derivatives (including but not limited to a
fusion
protein) or chemically modified derivatives of leptin across the blood-brain
barrier. A
fusion protein refers to a protein comprising a leptin polypeptide and a
different
1 S protein. The methods of the invention allow the enhancement of the
transport of
either endogenous leptin or exogenous leptin (including analogs, fragments,
consensus leptin, chemical derivatives thereof or fusion protein) across the
blood-
brain barrier. Conversely, in another embodiment of the invention, an
adrenergic
antagonist may be used to inhibit leptin transport across the blood-brain
barrier. An
exemplary adrenergic antagonist which acts to inhibit leptin transport into
the brain
thereby resulting in an increase in body weight includes but is not limited to
benoxathian, may be administered to an individual to increase body weight.
According to the present invention both purinergic and glutaminergic agonists
may
also be used to modulate leptin transport into the brain. An exemplary
purinergic
agonist comprises adenosine while an exemplary glutaminergic agonist comprises
glutamate.
Routes of administration of the compositions useful in the practice of
the invention include but are not limited to intravenous, intraarterial,
intraperitoneal,
intramuscular, intradermal, topical, intraocular, subcutaneous, intranasal,
oral,
intracisternal, intracerebroventricular, intrathecal, topical, intradermal, or
pulmonary.
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Conversely, in another preferred embodiment, an adrenergic antagonist may be
used
to inhibit leptin transport across the blood-brain barrier.
In one embodiment of the present invention, the composition
comprises one or more compounds selected from the group consisting of
adrenergic
agonists such as, but not limited to, epinephrine, isoproterenol, arterenol,
cirazoline,
phenylethylamine, epinephrine, norepinephrine, dopamine, nordefrin,
protokylol,
metaproterenol, metaraminol, phenylehprine, tyramine, hydroxyamphetamine,
nylidrin, isoxsuprine, methoxyphenamine, methoxamine, amphetamine,
methamphetamine, ephedrine, phenylpropanolamine, mephentermine,
chlorphentermine, tuaminoheptane, cyclopentamine, propylhexedrine, and analogs
and derivatives or metabolites thereof and optionally, a pharmaceutically
acceptable
Garner, excipient or diluent. Exemplary adrenergic antagonists includes, but
are not
limited to, phentolamine, prazosin, benoxathian, phenotybenzamine, and related
laloallyl-aminos.
In another embodiment, the compositions of the invention comprise
purinergic and glutaminergic agonists or combinations thereof and their use in
the
methods ofthe invention. Adenosine activates the adenosine, or purinergic 1
(P1)
receptor. P1 receptors are widespread in the body including the
cardiovascular,
respiratory, immune, and nervous systems. Adenosine blocks opioid-induced
feeding
and caffeine is a P 1 antagonist.
Glutamate is the endogenous ligand for glutamate (glutaminergic)
receptors. Glutamate receptors include ionotropic receptors (AMPA, kainate,
and N-
methyl-D-aspartate receptors), which directly control ion channels, and
metabotropic
receptors which act through second messenger systems. Glutamate receptors are
the
most common mediators of fast excitatory synaptic transmission in the central
nervous system. They are implicated in the mechanisms of memory and feeding.
Other compounds that affect feeding, suppress appetite, induce
anorexia, stimulate appetite, affect weight, or alter metabolism and which may
ultimately affect leptin transport across the blood-brain barrier and which
are useful in
the practice of the present invention include free fatty acids, sugars such as
glucose,
cytokines, drugs such as amphetamines, calcium channel Mockers, monoamines,
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amino acids, hormones including steroid hormones, dietary supplements,
ketones,
starches, micronutrients, lipoproteins, prostaglandins, prostacyclins,
peptides,
proteins, regulators of nitric oxide production, NMDA and GABA agonists and
antagonists, vitamins, minerals, and melatonin, and their precursors and
metabolites.
Preferred cytokines useful in the practice of the present invention
include, but are not limited to, interleukin 1 a, interleukin 1 (3,
interleukin 1 receptor
antagonist, interleukin 2, interleukin 6, interleukin 12, macrophage colony
stimulating
factor, macrophage inflammatory peptides such as MIP-1 a, MIP-1 ~3, and tumor
necrosis factor a (TNFa).
Other compounds used in the practice of the present invention include
fenflurimine and related compounds.
Other peptides and proteins useful in the practice of the present
invention either alone or in combination with other compounds described herein
include, but are not limited to, adrenocorticotropin hormone (ACTH), amylin,
atrial
natriuretic peptide (ANP), bombesin, calcitonin, calcitonin gene related
peptide
(CGRP), caerulein, cocaine and amphetamine regulated transcript peptide
(CART),
cholecystkinins (CCK), corticotropin releasing hormone (CRH), Cyclo-His-Pro,
enterostatin, FMRF-amide, galanin, glucagon, glucagon-like peptide (GLP),
growth
hormone, growth hormone releasing hormone (GHRH), gonadotropin hormone
releasing hormone (GnRH or LHRH), insulin, insulin-like growth factors,
macrophage migration inhibiting factor, melanocyte stimulating hormone (MSH),
motilin, MSH-inhibitory peptide (MIF-1), nerve growth factor (NGF),
neuromedins,
neuropeptide Y (NPY), neurotensin, neurotrophins (NT-3, NT-4), opiate peptides
(endorphins, enkephalins, endomorphins, dynoorphins, kyotorphin), orexin,
oxytocin,
pancreatic polypeptide, parathyroid hormone (PTH), pituitary adenylate cyclase
activating polypeptides (PACAP), sauvagine, somatostatin, substance P, thyroid
stimulating hormone (TSH), thyrotropin releasing hormone (TRH), tyrosine MIF-
1,
vasoactive intestinal polypeptide, and vasopressins.
Other compositions used in the practice of the present invention
comprise any of the foregoing compositions in combination with one another
and/or
in combination with one or more of the leptins described herein.
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This invention is also directed to a method for treating obesity which
comprises enhancing the transport of leptin, leptin variants, analogs,
consensus
leptins, fragments, or leptin derivatives thereof across the blood-brain
barrier
according to any of the preceding aspects or embodiments.
In yet another embodiment of this invention, methods and
compositions for treating metabolic disorders including obesity, diabetes
mellitus,
including type I and type II diabetes and insulin-resistant pathologies which
comprise
enhancing the transport of leptin, leptin variants, analogs, consensus
leptins,
fragments, or derivatives thereof across the blood-brain barrier according to
any of the
preceding aspects or embodiments are provided.
Also within the scope of the present invention are pharmaceutical
compositions useful for modulating body weight, the composition comprising
leptin
comprising the amino acid sequence set out in SEQ B7 NO: 2 or 4, SEQ )D NO: 5
and
SEQ ID NO: 6, consensus leptins, variants, analogs, leptin fusion proteins,
chemically
modified derivatives of leptin, and fragments thereof, and one or more agents
selected
from the group consisting of adrenergic agonists, adrenergic antagonists,
neurotransmitters, peptide hormones, cytokines, amino acids, opiate peptides,
punnergic agomsts, punnergic antagonists, glutaminergic agonists and
glutaminergic
antagonists, and metabolites thereof.
The invention also includes compositions and methods for modulating
body weight and/or treating metabolic disorders by modulating the regulatory
pathways which control appetite and/or metabolism. Because leptin appears to
play a
controlling role in appetite regulation, the methods and compositions of the
invention
are useful for modulating regulatory pathways in which the leptin plays a
role, perhaps
ultimately by regulating the transport of leptin across the blood-brain
barner.
The invention also comprises the use of adrenergic agonists, adrenergic
antagonists, neurotransmitters, peptide hormones, cytokines, amino acids,
opiate
peptides, purinergic agonists or antagonists, glutaminergic agonists or
antagonists, or
metabolites thereof for the manufacture of a medicament for modulating leptin
transport into the brain and/or for modulating body weight and/or for
modulating
appetite in a mammal. The uses may further comprise the use of any of the
leptins
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within the scope of the invention for the manufacture of the medicament for
modulating the transport of leptin across the blood-brain barrier and/or for
modulating
the body weight of a mammal. Preferred mammals for the practice of the present
invention are humans.
DETAILED DESCRIPTION OF THE INVENTION
The mammalian brain plays a central role in regulating the amount of
fat in a mammal in part by regulating food intake, food selection, and
thermogenesis.
The brain senses the fat level (adiposity) of the organism by sensing the
amount of
leptin in the blood of the organism which is transported into the brain via a
specific
saturable leptin transporter located at the blood brain barner. Obesity can
occur when
the brain incorrectly senses less than the appropriate amount of leptin in the
organism
which thereby triggers mechanisms to increase adiposity (e.g., increasing
feeding,
decreasing metabolic rate). Adiposity then increases until the brain senses an
appropriate level of leptin. It, therefore, follows that increasing the
efficiency of
transporting leptin across the blood brain barrier would be an effective way
to reduce
adiposity by increasing the amount of leptin effectively sensed by the brain.
Evidence suggests that the transporter responsible for leptin transport
across the blood-brain barrier is associated with or contains within its
structure sites
that when actuated modify the rate of leptin transport. These transporter rate
modifying sites are conceptually analogous to co-factor and/or allesteric
regulatory
sites for enzymes or co-factors. The presence of such sites therefore provide
attractive
therapeutic targets that can be used to regulate the transport of leptin
across the blood-
brain barrier thereby regulating adiposity in the mammal.
The present invention provides compositions and methods for
modulating body weight by modulating the signaling pathways involved in weight
regulation and/or appetite regulation. The invention also provides
compositions and
methods for modulating the transport of leptin across the blood-brain barrier
and
materials and methods for modulating appetite.
More particularly, the present invention is directed to compositions
including pharmaceutical compositions and methods for enhancing or inhibiting
the
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transport of leptin (0B) polypeptides across the blood-brain barrier. Such
methods
and compositions are useful in controlling the body weight of mammals,
including
humans. The methods and compositions are also useful in the treatment of
metabolic
disorders including diabetes mellitus (type I and type II). The compositions
and
methods of the present invention exploit the central role of leptin in the
regulation of
appetite and metabolism by modulating the transport of leptin across the blood-
brain
barrier to a site of action in the brain.
For the purposes of this invention, leptin and OB are used
interchangeably and refer to a polypeptide having as a mature form about 146
amino
acids.
Any leptin molecule, including leptin variants, analogs, fragments,
consensus leptins, or derivatives, which have the ability to modulate weight,
or to
alter metabolism in a host mammal, is useful in the practice of the present
invention.
Preferred leptin proteins useful in the practice of the present invention may
be native
murine leptin set out as SEQ >D NO: 2 which includes its signal sequence, or
its
mature form beginning at amino acid 21 (as numbered in SEQ ID NO: 2) of native
leptin and set out as SEQ 1D NO: 5 or protein as set forth in Zhang et al.
(Nature,
supra, herein incorporated by reference) or the native human OB protein (SEQ
1D
NO: 4) or its mature sequence beginning amino acids 21 through 166 set out as
SEQ
>D NO: 6. (See Zhang et al., Nature supra, at page 428.) Variants or analogs
of the
leptin proteins useful in the practice of the present invention include those
having a
substitution of one or more of its amino acids with another while still
maintaining a
biological activity of leptin. Natural variants of either leptin which lack a
glutamine
residue at position 28 of the mature sequence or other natural variants are
also useful
in the practice of the invention. Another example of a human leptin useful in
the
practice of the invention is an analog of SEQ ID NO: 6, which comprises 1 ) an
arginine in place of lysine at position 35; and 2) a leucine in place of
isoleucine at
position 74. (A shorthand abbreviation for this analog is the recombinant
human R-
>K~', L->I''~). The leptin molecules useful in the practice of the present
invention may
also optionally comprise a methionine at the N-terminus (-1 position).
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The murine leptin protein has significant homology to the human
protein, particularly as a mature protein, and, further, particularly at the N-
terminus.
One may prepare an analog of the recombinant human protein for use in the
practice
of the present invention by altering (such as substituting amino acid
residues), in the
recombinant human sequence, the amino acids which diverge from the murine
sequence. For example, using a human protein having a lysine at residue 35 and
an
isoleucine at residue 74 according to the numbering of SEQ ID NO: 6, wherein
the
first amino acid is valine, and the amino acid at position 146 is cysteine,
one may
substitute with another amino acid one or more of the amino acids at positions
32, 35,
50, 64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111, 118, 136,
138, 142,
and 145. One may select the amino acid at the corresponding position of the
murine
protein, (SEQ ID NO: 6), or another amino acid.
One may further prepare "consensus" molecules (consensus leptin or
consensus OB) based on the rat OB protein sequence [Murakami et al., Biochem.
Biophys. Res. Comm. 209:944-952 (1995) herein incorporated by referenceJ. Rat
OB
protein differs from human OB protein at the following positions (using the
numbering of SEQ >D NO: 6): 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97,
100, 101,
102, 105, 106, 107, 108, 111, 118, 136, 138, and 145. One may substitute with
another amino acid one or more of the amino acids at these divergent
positions. The
positions underlined and in bold print are those in which the murine leptin
protein as
well as the rat OB protein are divergent from the human OB protein, and thus,
are
particularly suitable for alteration. At one or more of these positions, one
may
substitute an amino acid from the corresponding rat OB protein, or another
amino
acid.
The positions from both mature rat and mature murine OB protein
which diverge from the mature human leptin protein, are: 4, 32, 33, 35, 50,
64, 68, 71,
74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 118, 136, 138,
142, and
145. A human OB protein according to SEQ >T7 NO: 6 having one or more of the
above amino acids deleted or replaced with another amino acid such as the
amino acid
found in the corresponding rat or murine sequence may also be effective.
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In addition, the amino acids found in rhesus monkey leptin protein
which diverge from the mature human OB protein are (with identities noted in
parentheses in one letter amino acid abbreviation): 8(S), 35(R), 48(V), 53(Q),
60(I),
66(I), 67(N), 68(L), 89(L), 100(L), 108(E), 112(D), and 118(L). Since the
recombinant human OB protein is active in cynomolgus monkeys, a human OB
protein according to SEQ ID NO: 4 or 6 having one or more of the rhesus monkey
divergent amino acids replaced with another amino acid, such as the amino
acids in
parentheses, may be effective. It should be noted that certain rhesus
divergent amino
acids are also those found in the above murine species (positions 35, 68, 89,
108, and
118). Thus, one may prepare a murine/rat/rhesus/human consensus molecule
(using
the numbering of SEQ >D NO: 6 having one or more of the amino acids at
positions
replaced by another amino acid: 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67,
68, 71, 74,
77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138,
142, and
145. The positions underlined and in bold print are those in which all three
species
are divergent from the human OB protein, and thus, are particularly suitable
for
alteration.
Other analogs may be prepared by deleting a part of the protein amino
acid sequence which results in a fragment of a leptin polypeptide. For
example, the
mature protein lacks a leader sequence which corresponds to amino acids 1-21
of SEQ
ID NO: 4. One may prepare the following truncated forms of the native human
leptin
protein molecules (using the number of SEQ >D NO: 6):
(a) amino acids 98-146
(b) amino acids 1-32
(c) amino acids 40-116
(d) amino acids 1-99 and (connected to) 112-146
(e) amino acids 1-99 and (connected to) 112-146 having one or
more of amino acids 100-111 placed between amino acids 99 and 112.
Also, the truncated forms (fragments) may also have altered one or
more of the amino acids which are divergent (in the rhesus, rat or murine OB
protein)
from human OB protein. Furthermore, any alterations may be in the form of
altered
amino acids, such as peptidomimetics or D-amino acids. Further, leptin
molecules
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having 83% or more amino acid identity with leptins having the amino acid
sequence
set out in SEQ ID NOs: 2, 4, ~ or 6 may also be used in the practice of the
invention.
Any of the foregoing leptin molecules may optionally have an N-
terminal methionine.
Also included with the scope of the invention are leptins encoded by
any of the polynucleotides set out in U.S. Patent No. 5,935,810 or any of the
polypeptides set out in U.S. Patent No. 6,001,968 which are incorporated by
reference
in their entirety.
The present protein (herein the term "protein" is used to include
"peptide" and OB analogs, such as those recited above, unless otherwise
indicated)
may also be derivatized by the attachment of one or more chemical moieties to
the
protein moiety. The chemically modified derivatives may be further formulated
for
intraarterial, intraperitoneal, intramuscular, subcutaneous, intravenous,
oral, nasal,
pulmonary, topical, ocular, intracisternal, intrathecal, transdermal,
intracerebroventricular, or other routes of administration. Chemical
modification of
biologically active proteins has been found to provide additional advantages
under
certain circumstances, such as increasing the stability and circulation time
of the
therapeutic protein and decreasing immunogenicity. See U.S. Patent No.
4,179,337,
Davis et al., issued December 18, 1979. For a review, see Abuchowski et al.,
Enzvmes as Drugs (J.S. Holcenberg and J. Roberts, eds. pp. 367-383 (1981)). A
review article describing protein modification and fusion proteins is Francis,
Focus on
Growth Factors 3:4-10 (May 1992) (published by Mediscript, Mountview Court ,
Friern Barnet Lane, London N20, OLD, UK).
The chemical moieties suitable for derivatization may be selected from
among various water soluble polymers. The polymer selected should be water
soluble
so that the protein to which it is attached does not precipitate in an aqueous
environment, such as a physiological environment. Preferably, for therapeutic
use of
the end-product preparation, the polymer will be pharmaceutically acceptable.
One
skilled in the art will be able to select the desired polymer based on such
considerations as whether the polymer/protein conjugate will be used
therapeutically,
and if so, the desired dosage, circulation time, resistance to proteolysis,
and other
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considerations. For the present proteins and peptides, the effectiveness of
the
derivatization may be ascertained by administering the derivative, in the
desired form
(i.e., by osmotic pump, or, more preferably, by injection or infusion, or,
further
formulated for oral, pulmonary or nasal delivery, for example), and observing
biological effects as described herein.
The water soluble polymer may be selected from the group consisting
of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene
glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrolidone, poly-
l, 3-
dioxolane, poly-l, 3, 6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random or non-random copolymers), and
dextran or poly (n-vinyl pyrolidone) polyethylene oxide/ethylene oxide co-
polymers,
polyoxyethylated polyols, polystyrenemaleate and polyvinyl alcohol.
Polyethylene
glycol propionaldenhyde may have advantages in manufacturing due to its
stability in
water.
Fusion proteins may be prepared by attaching polyaminoacids to the
OB protein (or analog) moiety. For example, the polyaminoacid may be a carrier
protein which serves to increase the circulatory half life of the protein. For
the
present therapeutic or cosmetic purposes, such polyaminoacid should be those
which
do not create neutralizing antibody response, or other adverse response. Such
polyaminoacid may be selected from the group consisting of serum albumin (such
as
human serum albumin), an antibody or portion thereof (such as an antibody
constant
region, sometimes called "F~") or other polyaminoacids. As indicated below,
the
location of attachment of the polyamino acid may be at the N-terminus of the
OB
protein moiety, or other place, and also may be connected by a chemical
"linker"
moiety to the OB protein.
In the case of an OB-Fc fusion, the OB is typically fused at its C-
terminus with the N-terminus. However, OB may be fused at its N-terminus with
the
C-terminus of the Fc molecule. Typically, in such fusions, the fused protein
will
retain at least functionally active hinge CH2 and CH3 domains of the constant
region
of the immunoglobulin heavy chain. Fusions may also be made to the C-terminus
of
the Fc portion of a constant domain or immediately N-terminal to the CH1
domain of
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the heavy chain or the corresponding region of the light chain. The exact site
at which
the fusion is made is not critical. The fusion proteins may comprise multimers
of the
Fc-OB fusion.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 2 kDa and about 100 kDa (the term "about" indicating that in
preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic
protein or analog).
The number of polymer molecules so attached may vary, and one
skilled in the art will be able to ascertain the effect on function. One may
mono-
derivatize, or may provide for a di-, tri-, tetra- or some combination of
derivatization,
with the same or different chemical moieties (e.g., polymers, such as
different weights
of polyethylene glycols). The proportion of polymer molecules to protein (or
peptide)
molecules will vary, as will their concentrations in the reaction mixture. In
general,
the optimum ratio (in terms of efficiency of reaction in that there is no
excess
unreacted protein or polymer) will be determined by factors such as the
desired degree
of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the
polymer
selected, whether the polymer is branched or unbranched, and the reaction
conditions.
The chemical moieties should be attached to the protein with
consideration of effects on functional or antigenic domains of the protein.
There are a
number of attachment methods available to those skilled in the art. E~g., EP 0
401
384 herein incorporated by reference (coupling PEG to G-CSF), see also Malik ,
Exp.
Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound through
amino
acid residues via a reactive group, such as, a free amino or carboxyl group.
Reactive
groups are those to which an activated polyethylene glycol molecule may be
bound.
The amino acid residues having a free amino group may include lysine residues
and
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the N-terminal amino acid residue. Those having a free carboxyl group may
include
aspartic acid residues, glutamic acid residues, and the C-terminal amino acid
residue.
Sulfhydryl groups may also be used as reactive groups for attaching the
polyethylene
glycol molecule(s). Preferred for therapeutic purposes is attachment at an
amino
group, such as attachment at the N-terminus or lysine group. Attachment at
residues
important for receptor binding should be avoided if receptor binding is
desired.
One may specifically desire N-terminally chemically modified OB
protein or polypeptides. Using polyethylene glycol as an illustration of the
compositions useful in the practice of the present invention, one may select
from a
variety of polyethylene glycol molecules (by molecular weight, branching,
etc.), the
proportion of polyethylene glycol molecules to protein molecules in the
reaction mix,
the type of pegylation reaction to be performed, and the method of obtaining
the
selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties
if necessary) may be by purification of the N-terminally pegylated material
from a
population of pegylated protein molecules. Selective N-terminal chemical
modification may be accomplished by reductive alkylation which exploits
differential
reactivity of different types of primary amino groups (lysine versus the N-
terminal)
available for derivatization in a particular protein. Under the appropriate
reaction
conditions, substantially selective derivatization of the protein at the N-
terminus with
a carbonyl group containing polymer is achieved. For example, one may
selectively
N-terminally pegylate the protein by performing the reaction at a pH which
allows one
to take advantage of the pKa differences between the E-amino group of the
lysine
residues and that of the a-amino group of the N-terminal residue of the
protein. By
such selective derivatization, attachment of a water soluble polymer to a
protein is
controlled: the conjugation with the polymer takes place predominantly at the
N-
terminus of the protein and no significant modification of other reactive
groups, such
as the lysine side chain amino groups, occurs. Using reductive alkylation, the
water
soluble polymer may be of the type described above, and should have a single
reactive
aldehyde for coupling to the protein. Polyethylene glycol propionaldehyde,
containing
a single reactive aldehyde, may be used.
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An N-terminally monopegylated derivative is preferred for ease in
production of a therapeutic, N-terminal pegylation ensures a homogenous
product as
characterization of the product is simplified relative to di-, tri- or other
multi-
pegylated products. The use of the above reductive alkylation process for
preparation
of an N-terminal product is preferred for ease in commercial manufacturing.
As described in the following examples, the administration of
compositions which interact with an adrenoreceptor (preferably adrenergic
agonists)
either prior to or concurrently with the administration of leptin
significantly increases
the amount of leptin which crosses the blood-brain barrier into the brain. The
compositions and methods of the invention are also useful for increasing the
transport
of endogenous leptin across the blood-brain barner. These results are
illustrated by
the following Examples in which radiolabelled leptin was administered then
measured
in mice who were given compositions which interact with an adrenoreceptor.
Compositions containing epinephrine (which reacts with an adrenoreceptor) were
the
most effective in enhancing leptin transport. Other compositions including
those
containing amino acids or hormones were tested as well and in some cases were
shown to be effective in enhancing leptin transport across the blood-brain
barner.
The invention is described in the following examples by way of
illustration and should not be construed as limiting the invention as set out
in the
appended claims.
Example 1 describes the effects of epinephrine on leptin transport
across the blood-brain barrier.
Example 2 describes the effect of various dosages of epinephrine on
the transport of leptin across the blood-brain barrier.
In Example 3, the effect of epinephrine on the integrity of the blood-
brain barrier was examined.
In Example 4, the effect of the amino acids tyrosine and phenylalanine
on transport of leptin across the blood-brain barner was studied.
In Example 5, the effect of arginine, phenylalanine, tryptophan, and
tyrosine on the transport of leptin across the blood-brain barrier was
studied.
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In Example 6, the effects of neurotransmitters including dopamine,
histamine, serotonin, and epinephrine on leptin transport are described.
Example 7 describes the effect of co-administration of the
adrenoreceptor agonists/antagonists cirazoline hydrochloride, UK14304,
albuterol,
CGP-12177A, and benoxathian hydrochloride on transport of leptin across the
blood-
brain barner was examined.
In Example 8, the effect of co-administration of certain adrenoreceptor
agonists such as isoproterenol, clonidine, arterenol, and phenylephrine on
transport of
leptin across the blood-brain barner was examined.
In Example 9, the effect of the adrenoreceptor antagonists
phentolamine, D,L-propanolol, yohimbine, and prasozin on transport of leptin
across
the blood-brain barrier was tested.
Example 10 describes the effect of tumor necrosis factor on leptin
transport across the blood-brain barrier.
Example 11 describes the effect of purinergic and glutaminergic
agonists on the transport of leptin across the blood-brain barrier.
EXAMPLE 1
Effects of Administration of EEinephrine on Transport of Leptin
across the Blood-Brain Barner in Mice
In this Example, the effect of administering epinephrine on the
transport of leptin across the blood-brain barner in mice was studied.
In these experiments, six groups of five male ICR mice (Blue Spruce
Farms, Altamont, NY) weighing about 17-22 g were anaesthetized with ethyl
carbamate (4 g/kg) then had their jugular vein and carotid artery surgically
exposed.
The mice were then given an intraperitoneal (i.p.) injection of epinephrine
(33 Itg/200
p1) in lactated Ringer's solution with 1 % bovine serum albumin. The time of
these
injections was considered time zero. After time intervals of 10 minutes
(min.), 30
min., 45 min., 1 hour (h), and 2 h post epinephrine injection, radiolabelled
leptin ('25I,
1.65 x 106 cpm) in lactated Ringer's solution with 1% bovine serum albumin was
administered to the mice via intravenous (i.v.) injection in the jugular vein.
The
control mice were not given epinephrine, only lactated Ringer's solution with
1%
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bovine serum albumin and were injected with '-'~I- leptin only after the time
interval of
minutes. All mice were decapitated and their blood collected after 10 minutes
following the leptin injection. The brain (except pituitary and pineals) was
removed
and counted in a gamma counter (Micromedic 4/200, Horsham, PA) for 3 minutes.
5 Blood was collected from a cut in the right carotid artery, centrifuged at
2000 g for 10
min. at 4°C, then 0.1 ml was counted in a gamma counter. Brain/blood
ratios were
expressed as counts algebraically to pl/g of brain over counts/min./pl of
arterial blood.
Table 1. Administration of Epinephrine Followed by Leptin
Time post epinephrine administration'-'SI-leptin, brain/serum
~ std. dev.
(cpm/g)/(cpm/ p l)
10 control (10 min., no epinephrine)15.68 t 2.28
10 min. 23.69 t 3.90
30 min. 23.27 t 3.45
45 min. 18.68 ~ 2.19
1 h 20.73 ~ 2.68
2 h 24.86 ~ 4.02
The results of this experiment indicate that the administration of
epinephrine prior to the administration of leptin enhances the uptake of
leptin by the
brain in mice. More specifically, in the control mice who received no
epinephrine, the
amount of radiolabelled leptin in the brain following its i.v. administration
was
15.68~2.28 counts/min./g of brain over counts/min./pl of arterial blood as
compared
to the amount of leptin in the mice who received epinephrine prior to leptin
administration was 23.69~3.90 counts/min./g of brain over counts/min./pl of
arterial
blood. This represents an enhancement in leptin uptake by the brain of
approximately
51 %. The other time points of 30 min., 45 min., 1 h, and 2 h illustrate that
epinephrine still exerts its positive effects on leptin uptake by the brain
even after a
time interval of 2 h has passed.
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EXAMPLE 2
Effects of Administration of Epinephrine on Blood-Brain Barrier in Mice
In this Example, the effect of various dosages of epinephrine on the
transport of leptin across the blood-brain barrier was studied.
S As in Example l, mice were anaesthetized with ethyl carbamate. The
mice were then given an i.v. injection of a solution containing radiolabelled
leptin
('Z'I, 2.1 x 106 cpm) in lactated Ringer's solution with 1% bovine serum
albumin and
various amounts of epinephrine (133.33 pg, 400 nM; 66.6 pg, 200 nM; 33.3 pg,
100
nM; 13.3 pg, 40 nM; 0.667 pg, 2 nM) in 200 p1. Blood and brain samples were
collected as described in the previous Examples at 10 min post leptin
injection.
Table 2. Co-administration of Epinephrine with Leptin
Time post epinephrine '25I, brain/blood t std. dev.
administration (10 min.) (cpm/g)/(cpm/ p 1)
control (no epinephrine) 24.54 t 5.6
+ epinephrine 2 nM 28.68 ~ 10.7
+ epinephrine 40 nM 62.73 ~ 27.9
+ epinephrine 100 nM 74.18 t 20.3
+ epinephrine 200 nM 64.75 + 21.3
+ epinephrine 400 nM all mice died instantly
The results of this experiment indicate that the co-administration of the
leptin plus epinephrine enhances the uptake of leptin by the brain. Likewise,
at 40 nM
epinephrine the uptake was increased by about 155%, at 100 nM epinephrine the
uptake increased by about 200%, at 200 nM epinephrine the uptake was increased
about 163% but two of the five mice in that group died, and at 400 nM
epinephrine all
of the mice died. These data were corrected for the amount of residual blood
in the
brain after removal of capillaries by gradient centrifugation.
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EXAMPLE 3
Effects of Administration of Epinephrine on the
lnte r~ity of the Blood-Brain Barrier in Mice
In this Example, the effects of the administration of epinephrine on the
S integrity of the blood-brain barrier in mice was evaluated. Radiolabelled
albumin is
the traditional standard to be administered and monitored in order to test the
integrity
of the blood-brain barrier (Davson. H. ( 1967) Physiology of the Cerebrospinal
Fluid,
pp. 82-103, J. & A. Churchill, London).
As described above, mice were anaesthetized with ethyl carbamate
then given an i.v. injection of either a solution containing radiolabelled
leptin ('25I,
1.54 x 106 cpm) and albumin (99Tc, 3.4 x 106 cpm) (labeled solution) in
lactated
Ringer's solution with 1 % bovine serum albumin in 200 p1 or the labeled
solution
plus epinephrine (33 pg). All mice were decapitated with their blood and
testis
collected after 10 minutes following the leptin injection. The brain (except
pituitary
and pineals) was removed and counted in a gamma counter (Micromedic 4/200,
Horsham, PA) for 3 minutes. Blood was also collected from a cut in the right
carotid
artery, centrifuged at 2000 g for 10 min. at 4°C, then 50 p1 was
counted in a gamma
counter. Brain/blood and testis/blood ratios were expressed as counts/min./g
of brain
or testis over counts/min./pl of arterial blood.
Table 3. Co-administration of Epinephrine with Leptin and Albumin
Time post brain/blood.brain/bloodtestis/bloodtestis/blood
epinephrine (CDTri/~l (cnm/g~
administration(cpm/ p l) (cpm/ p (cpm/ p (cpm/ p
1) 1) 1)
control (no 'z'I-leptin 99Tc-albumin'z5I-leptin99Tc-albumin
epinephrine)
1 min. 12.57 10.02 8.03 4.20
2 min. 15.66 10.22 7.71 4.52
3 min. 16.80 9.87 10.64 4.45
4 min. 22.18 10.14 23.76 8.23
5 min. 22.84 10.85 23.92 8.85
7.5 min. 23.46 10.38 29.55 9.06
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Time post brain/blood.brain/blood testis/bloodtestis/blood
epinephrine c m/~ (CDII1/~) ~cpm/e)
administration(cpm/ p (cpm/ p 1) (cpm/ p (cpm/ ~
1) I) I)
min. 27.48 11.60 39.02 10.74
12.5 min. 24.36 10.47 44.36 14.42
+40nM
epinephrine
5 1 min. 19.50 10.38 5.37 2.90
2 min. 22.11 11.28 9.55 4.70
3 min. 27.51 10.84 9.98 4.09
4 min. 36.82 10.46 18.42 6.56
5 min. 28.97 10.62 29.72 7.78
10 7.5 min. 36.55 10.27 58.68 17.52
10 min. 38.38 11.82 25.38 8.42
12.5 min. 50.53 10.54 152.38 34.09
The results of this experiment indicate that the co-administration of
epinephrine with leptin induced an enhanced uptake of leptin by the brain in
mice.
The data from the time points of 1 min., 2 min., 3 min., 4 min., 5 min., 7.5
min., 10
min., and 12 min. illustrate that the effects of epinephrine on leptin uptake
by the
brain increase with time as shown in this 12 minute experiment. The co-
administration of epinephrine did not enhance the uptake of albumin in the
brain of
mice. This shows that the increased uptake of leptin by the brain when
epinephrine is
administered is not the result of a damaged blood-brain barner, as the amount
of 99Tc-
albumin crossing the blood-brain barrier remains nearly the same in the
presence or
absence of epinephrine. In other studies, epinephrine was shown to increase
the
uptake of both leptin and albumin by testis. While those data indicate that
epinephrine may act by disrupting the blood testis barner, they nonetheless
provide
evidence that leptin uptake in tissues other than the brain may be enhanced
using the
cytokines, peptides, neurotransmitters and other molecules according to the
present
invention.
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EXAMPLE 4
Effects of Administration of Amino Acids on Transport
of Leptin Across the Blood-Brain Barrier in Mice
In this Example, the effect of administration of various amino acids on
transport of leptin across the blood-brain barner was studied.
As in the prior Examples, mice were anaesthetized with ethyl
carbamate. The mice were then given an i.v. injection of either a labeled
solution
containing radiolabelled human leptin ('25I, 1.58 x 106 cpm) in lactated
Ringer's
solution with 1 % bovine serum albumin in 200 p1 or the labeled solution plus
one of
the following amino acids (tyrosine or phenylalanine, 10 ug). Blood and brain
samples were collected as described in the previous Examples at the following
time
points post leptin injection (1 min., 2 min., 3 min., 4 min., 5 min., 7.5
min., 10 min.,
12.5 min. and 15 min.).
Table 4. Co-administration of Amino Acids with Leptin
Time post amino acid administrationbrain/blood
(cpm/g)/(cpm/ p 1)
control (no amino acid) 'Z5I-leptin
1 min. 12.98
2 min. 16.34
3 min. 15.64
4 min. 16.71
5 min. 18.38
7.5 min. 17.42
10 min. 19.00
12.5 min. 22.83
15 min. 26.33
+ tyrosine
1 min. 13.34
2 min. 14.67
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Time post amino acid administrationbrainlblood
(cpm/g)/(cpm/ p 1)
3 min. 15.04
4 min. 16.35
min. 17.55
7.5 min. 18.26
5 10 min. 22.90
12.5 min. 24.11
min. 23.86
+ phenylalanine
1 min. 12.24
10 4 min. 16.15
5 min. 14.39
10 min. 16.67
12.5 min. 20.64
15 min. 23.55
15 The results of this experiment indicate that the co-administration of
tyrosine with leptin enhanced the uptake of leptin by the brain. However,
phenylalanine had no such effect. The enhancement of leptin uptake by tyrosine
was
time dependent over the tested interval of 15 minutes.
EXAMPLE 5
Effects of Administration of Other Amino Acids on Transport
of Le~tin Across the Blood-Brain Barrier in Mice
In this Example, the effect of other amino acids on the transport of
leptin across the blood-brain barrier was studied.
As in the prior Examples, mice were anaesthetized with ethyl
carbamate. The mice were then given an i.v. injection of either a solution
containing
radiolabelled leptin (''5I, 1.68 x 1O6 cpm) in lactated Ringer's solution with
1% bovine
serum albumin in 200 p1 or the solution plus one of the following amino acids
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(arginine, phenylalanine, tryptophan, or tyrosine, 1 mg). Blood and brain
samples
were collected as described in the previous Examples at 10 min post leptin
injection.
Table 5. Co-administration of Amino Acids with Leptin
Time post leptin administration''SI-leptin, brain/blood
( 10 min.) ~ std. dev.
(cP~~)~(cp~ N 1)
control (no epinephrine) 15.30 ~ 5.05
+ arginine 18.82 t 2.58
+ phenylalanine 19.83 ~ 5.29
+ tryptophan 17.97 ~ 4.33
+ tyrosine 23.73 t 8.84
The results of this experiment indicate that the co-administration of
arginine, phenylalanine, or tryptophan with leptin did not affect the uptake
of leptin by
the brain. However, the administration of tyrosine significantly enhanced
leptin
uptake by the brain. Similar studies also showed that neither leucine,
threonine, nor
glycine had an effect on transport of leptin across the blood-brain barner.
EXAMPLE 6
Effects of Administration of Neurotransmitters on Transport
of Leptin Across the Blood-Brain Barner in Mice
In this Example the effect of certain neurotransmitters on the transport
of leptin across the blood-brain barrier was studied.
As described above, groups of mice were anaesthetized with ethyl
carbamate. The mice were then given by intracerebroventricular (icy) injection
of a
solution containing a neurotransmitter such as: acetylcholine, 98 pg;
dopamine, 103
lxg; epinephrine, 55 pg; histamine, 117 fig; or serotonin, 130 pg. Ten minutes
later
the mice were given an i.v. injection containing radiolabelled leptin ('-''I,
1.77 x 106
cpm) in lactated Ringer's solution with 1 % bovine serum albumin in 100 u1.
Blood
and brain samples were collected as described in the previous Examples at 10
min
post leptin injection.
Table 6. Administration of Neurotransmitters with Leptin
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Time post leptin administration''-5I-leptin, brain/blood
( 10 min.) ~ std. dev.
(cpm/g)/(cpm/ ~ 1)
control (no epinephrine)12.95 ~ 3.31
+ acetylcholine 13.78 ~ 4.73
+ dopamine 13.17 ~ 2.94
+ epinephrine 15.37 ~ 3.12
+ histamine 10.85 ~ 4.22
+ serotonin 11.32 ~ 3.38
The results of this experiment indicate that the icv administration of
the neurotransmitters acetylcholine, dopamine, histamine, and serotonin with
leptin
had no effect on the uptake of leptin by the brain. The administration of
epinephrine
by the icv route prior to leptin also did not enhance the uptake of leptin by
the brain.
This shows that the site at which epinephrine acts to modify leptin transport
is on the
blood side of the blood-brain barner.
In another series of studies, neurotransmitters were injected
intravenously with 200 nmol/mouse of either acetylcholine, dopamine,
epinephrine,
histamine, or serotonin. The results of this study indicate that only
epinephrine was
capable of enhancing transport of leptin across the blood-brain barrier.
EXAMPLE 7
Effects of Administration of Adrenorec~tor A~onists on
Transport of Leptin Across the Blood-Brain Barrier in Mice
In this Example, the effect of certain adrenoreceptor agonists on the
transport of leptin across the blood-brain barrier was studied.
As in the prior Examples, mice were anaesthetized with ethyl
carbamate. The mice were then given an i.v. injection of either a labeled
solution
containing radiolabelled leptin ('ZSI, 1.98 x 106 cpm) in lactated Ringer's
solution with
1 % bovine serum albumin in 100 ~1 or the labeled solution plus one of the
following
agonists: isoproterenol, 25.30 pg; clonidine, 18.66 gg; epinephrine, 14.26
fig; L-
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phenylephrine, 14.26 pg; or arterenol, 22.35 pg). Blood and brain samples were
collected as described above at 10 min post leptin injection.
Table 7. Co-administration of Adrenoreceptor Agonists with Leptin
Time post administration 'z5I-leptin, brain/blood ~
(10 min.) std. dev.
(cpm/g)/(cpm/ p 1)
control (no agonist) 14.07 ~ 1.88
+ isoproterenol 20.61 t 4.05
+ clonidine 15.05 t 1.54
+ arterenol (norepinephrine)23.78 ~ 6.35
+ L-phenylephrine 19.19 t 4.57
The results of this experiment indicate that the co-administration of the
adrenoreceptor agonists, isoproterenol and arterenol with leptin enhanced the
uptake
of leptin by the brain. However, clonidine and L-phenylephrine had no effect
on
leptin transport
EXAMPLE 8
Effects of Administration of Adrenergic Antagonists on
Transport of Leptin Across the Blood-Brain Barrier in Mice
In this Example, the effect of the co-administration of epinephrine with
adrenoreceptor antagonists on transport of leptin across the blood-brain
barrier was
studied.
As described above, mice were anaesthetized with ethyl carbamate.
The mice were then given an i.v. injection of either a labeled solution
containing
radiolabelled leptin ('25I, 1.48 x 106 cpm) in lactated Ringer's solution with
1% bovine
serum albumin and epinephrine (3.33 pg) in 100 p1, or labeled leptin and
epinephrine
plus one of the following antagonists (phentolamine, 528.4 pg; D,L-propanolol,
4.41
pg; yohimbine, 547.3 pg; or prazosin, saturated solution of 587.8 pg). Blood
and
brain samples were collected as described above at 10 min post leptin
injection.
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Table 8. Co-administration of Adrenoreceptor Antagonists and Epinephrine with
Leptin
Time post epinephrine ''SI-leptin, brain/blood t
administration ( 10 min.)std. dev.
(cpm/g)/(cpm/ ~ 1)
control (epinephrine 26.78 ~ 4.62
alone)
+ phentolamine 18.86 t 4.27
+ D, L-propanolol 25.68 ~ 4.02
+ yohimbine 17.38 ~ 3.30
+ prazosin 18.06 ~ I .89
The results of this experiment indicate that the co-administration of the
adrenoreceptor antagonists plus epinephrine either had no effect or reduced
the uptake
of leptin by the brain. Specifically, D,L-propanolol (a ~3 antagonist) had no
effect
while phentolamine, yohimbine, and prasozin (a antagonists) had a negative
effect as
compared to the control.
EXAMPLE 9
Effects of Administration of Adrener~ic Agonists/Antagonists on
Transport of Leptin Across the Blood-Brain Barrier in Mice
In this Example, the effect of adrenoreceptor agonists and antagonists
on the transport of leptin across the blood-brain barner was studied.
As in the prior Examples, mice were anaesthetized with ethyl
carbamate. The mice were then given an i.v. injection of either a solution
containing
radiolabelled leptin ('25I, 1.2 x 106 cpm) in lactated Ringer's solution with
1% bovine
serum albumin in 100 p l or the labeled solution plus one of the following
agonists
(cirazoline hydrochloride, 25 pg; albuterol, 50 fig; UK 14304; epinephrine,
13.3 pg)
or antagonists (benoxathian hydrochloride, 250 pg + epinephrine, 13.3 pg; CGP-
12177A (250 pg) and epinephrine, 13.3 pg). Blood and brain samples were
collected as described in the previous Examples at 10 min post leptin
injection.
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Table 9. Co-administration of a,, a,_ or ~3 Adrenoreceptor
Agonists/Antagonists with
Leptin
Time post epinephrine administration'--''I-leptin, brain/blood
( 10 min. ) ~ std. dev.
(cpm/g)/(cpm/ p 1)
S control (no epinephrine) 17.0 ~ 2.74
+ DMSO 25.1 ~ 2.13
+ epinephrine 47.1 ~ 17.3
+ cirazoline (a, agonist) 45.5 t 11.3
+DMSO + LJK14304 (a, agonist)14.7 + 3.98
+ albuterol ((3 agonist) 18.4 ~ 2.39
+ epinephrine + CGP 12177A 39.7 ~ 8.70
(~3
antagonist)
+ epinephrine + benoxathian19.7 ~ 5.01
(a, antagonist)
The results of this experiment indicate that the co-administration of the
a, agonist cirazoline with leptin enhanced the uptake of leptin by the brain.
The data
also show that the a, antagonist benoxathian blocked the enhancing effect of
epinephrine.
EXAMPLE 10
Modulating Leptin Transport by Tumor Necrosis Factor a (TNF-a~
TNF-a (cachexin) is a cytokine about the same size as leptin that is
transported across the blood-brain barrier and also has effects on feeding.
This
suggests the possibility that TNF may modulate the transport of leptin across
the
blood-brain barrier. Similarly, leptin may play a role in the transport of TNF
across
the blood-brain barrier. This hypothesis was tested using two experimental
paradigms, an acute model, and a chronic model.
Acute
This experiment determined whether the acute administration of TNF
would acutely affect the entry of radioactively labeled leptin (I-leptin) into
the brain.
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Three groups of mice were tested. One group received labeled leptin (described
in
Examples set out above) alone; a second group received labeled leptin plus 1
pg/mouse of mouse TNF; a third group received 1 pg /mouse of human TNF and
labeled leptin. After injection, brain and blood samples were obtained at
times similar
to those used in the Examples set out above over time. (It should be noted
that this is
a high dose of TNF.) There was no difference among these groups in the amount
of
labeled leptin transported into the mouse brain. These data show that TNF and
leptin
do not share the same transporter (no competition or inhibition of leptin
transport) nor
does it acutely upregulate the leptin transporter (no enhancement of leptin
transport).
Chronic
This experiment was performed in mice that were genetically altered so
that both of the receptors for TNF were "knocked out" and therefore did not
express
active TNF receptors. As such, these mice were insensitive to TNF. The rate of
uptake of labeled leptin in these mice was then determined in comparison to
controls.
The amount of unlabeled leptin that was needed to inhibit the leptin
transporter in
these mice was also determined.
The entry rate of 0.477 pl/g-min for labeled leptin is similar to that
typically found in normal mice. However, the entry of I-leptin into the brain
was not
inhibited by 0.1 or 0.3 pg/mouse of unlabeled leptin and 1.0 pg/mouse
inhibited the
entry rate by only 40%. In normal mice, 0.3 pg inhibits entry by SO% and 1
pg/mouse
inhibits entry by 95%.
These data suggest that TNF receptor knockout mice have an altered
transporter for leptin across the blood-brain barrier. Chronic TNF exposure,
perhaps
especially during development, is likely needed for the normal functioning of
the
leptin transporter. This, therefore, represents another class of compounds
(cytokines),
in addition to the adrenergic agonist, amino acids, and other compositions
described
above that can modulate the transport of leptin across the BBB.
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EXAMPLE 11
Modulation of Leptin Uptake by Adenosine and Glutamate
Studies were conducted to determine whether compounds which
interact with purinergic receptors or with glutamate (glutaminergic) receptors
are
capable of modulating the uptake of leptin into the brain.
In particular, these studies were conducted to determine whether the
acute administration of adenosine, arginine, or glutamate could affect the
entry of
radioactively labeled leptin (I-leptin) into the brain. Four groups of mice
were tested.
One group received labeled leptin (as described in examples set out above)
alone, a
second group received labeled leptin plus adenosine (0.4 mmol/kg), a third
group
received labeled leptin plus arginine (10 mg/mouse), and a fourth group
received
labeled leptin plus glutamate at the dosage of 10 mg/mouse.
L-Arginine is an essential amino acid included below as a control.
After injection, brain and blood samples were obtained at 10 min post leptin
injection
as described. The brain/serum ratios have been corrected for vascular space by
subtracting 10 ~.I/g.
Table 10. Co-administration of Adenosine, Arginine, or Glutamate with Leptin
Compound 'z5I-leptin" brain/blood
_+ std dev.
(cP~g) (cP~l~1)
I-leptin Only (Control) 21.6 + 1.96
Adenosine 14.9 + 4.79
Arginine 21.8 + 5.29
Glutamate 9.67 + 2.3
The results of this experiment shown in Table 10 indicate that
co-administration of the purinergic agonist adenosine or the glutamate agonist
significantly decreased the transport of leptin into the brain. Co-
administration of
arginines an essential amino acid had no effect on leptin transport.
The results set out above show that purinergic agonists such as
adenosine are useful in decreasing uptake of leptin into the brain and thus
may act as
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therapeutic agents in pathological conditions or under other circumstances
wherein a
decrease in leptin uptake in the brain is desired.
Similarly the results shown in Table 10 show that agonists (e.g.,
glutamate) that interact with glutamate receptors including the ionotropic
receptors
(e.g. AMPA, and N-methyl-D-aspartate receptors) and metabotropic receptors may
also be useful in the same context as the purinergic agonists described above.
While the present invention has been described in terms of specific
embodiments, it is understood that variations and modifications will occur to
those
skilled in the art. All of the foregoing references are hereby incorporated by
reference.
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SEQUENCE LISTING
<110> Banks, William A.
<120> MODULATIONTHEBLOOD-BRAIN BARRIER TRANSPORTER FOR
OF
LEPTIN
<130> 01017/35040
<140>
<141>
<160> 6
<170> PatentIn 2.0
Ver.
<210> 1
<211> 2793
<212> DNA
<213> Murine
<220>
<223> Murine cDNA
ob (leptin)
<220>
<221> CDS
<222> (57)..(557)
<220>
<221> sig peptide
<222> (57)..(59)
<220>
<221> mat~peptide
<222> (60)..(557)
<400> 1
ggatccctgc tccagcagct gatcccaggg 59
gcaaggtgca agaagaagaa aggaaa
atg
Met
-1
tgc tgg aga ccc tgtcgg ctgtggctttggtcc tatctgtct 107
ctg ttc
Cys Trp Arg Pro CysArg LeuTrpLeuTrpSer TyrLeuSer
Leu Phe
1 5 10 15
tat gtt caa gca cctatc aaagtccaggatgac accaaaacc 155
gtg cag
Tyr Val Gln Ala ProIle LysValGlnAspAsp ThrLysThr
Val Gln
20 25 30
ctc atc aag acc gtcacc atcaatgacatttca cacacgcag 203
att agg
Leu Ile Lys Thr ValThr IleAsnAspIleSer HisThrGln
Ile Arg
35 40 45
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tcg gta tcc gcc aag cag agg gtc act ggc ttg gac ttc att cct ggg 251
Ser Val Ser Ala Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly
50 55 60
ctt cac ccc att ctg agt ttg tcc aag atg gac cag act ctg gca gtc 299
Leu His Pro Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val
65 70 75 80
tat caa cag gtc ctc acc agc ctg cct tcc caa aat gtg ctg cag ata 347
Tyr Gln Gln Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile
85 90 95
gcc aat gac ctg gag aat ctc cga gac ctc ctc cat ctg ctg gcc ttc 395
Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe
s 100 105 110
tcc aag agc tgc tcc ctg cct cag acc agt ggc ctg cag aag cca gag 443
Ser Lys Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu
115 120 125
agc ctg gat ggc gtc ctg gaa gcc tca ctc tac tcc aca gag gtg gtg 491
Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val
130 135 140
get ttg agc agg ctg cag ggc tct ctg cag gac att ctt caa cag ttg 539
Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu
145 150 155 160
gat gtt agc cct gaa tgc tgaagtttca aaggccacca ggctcccaag 587
Asp Val Ser Pro Glu Cys
165
aatcatgtag agggaagaaa ccttggcttc caggggtctt caggagaaga gagccatgtg 647
cacacatcca tcattcattt ctctccctcc tgtagaccac ccatccaaag gcatgactcc 707
acaatgcttg actcaagtta tccacacaac ttcatgagca caaggagggg ccagcctgca 767
gaggggactc tcacctagtt cttcagcaag tagagataag agccatccca tcccctccat 827
gtcccacctg ctccgggtac atgttcctcc gtgggtacac gcttcgctgc ggcccaggag 887
aggtgaggta gggatgggta gagcctttgg gctgtctcag agtctttggg agcaccgtga 947
aggctgcatc cacacacagc tggaaactcc caagcagcac acgatggaag cacttattta
1007
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tttattctgc attctatttt ggatggatct gaagcaaggc atcagctttt tcaggctttg
1067
ggggtcagcc aggatgagga aggctcctgg ggtgctgctt tcaatcctat tgatgggtct
1127
gcccgaggca aacctaattt ttgagtgact ggaaggaagg ttgggatctt ccaaacaaga
1187
gtctatgcag gtagcgctca agattgacct ctggtgactg gttttgtttc tattgtgact
1247
gactctatcc aaacacgttt gcagcggcat tgccgggagc ataggctagg ttattatcaa
1307
aagcagatga attttgtcaa gtgtaatatg tatctatgtg cacctgaggg tagaggatgt
1367
gttagaggga gggtgaagga tccggaagtg ttctctgaat tacatatgtg tggtaggctt
1427
ttctgaaagg gtgaggcatt ttcttacctc tgtggccaca tagtgtggct ttgtgaaaag
1487
gacaaaggag ttgactcttt ccggaacatt tggagtgtac caggcaccct tggaggggct
1547
aaagctacag gccttttgtt ggcatattgc tgagctcagg gagtgagggc cccacatttg
1607
agacagtgag ccccaagaaa agggtccctg gtgtagatct ccaaggttgt ccagggttga
1667
tctcacaatg cgtttcttaa gcaggtagac gtttgcatgc caatatgtgg ttctcatctg
1727
attggttcat ccaaagtaga accctgtctc ccacccattc tgtggggagt tttgttccag
1787
tgggaatgag aaatcactta gcagatggtc ctgagccctg ggccagcact gctgaggaag
1847
tgccagggcc ccaggccagg ctgccagaat tgcccttcgg gctggaggat gaacaaaggg
1907
gcttgggttt ttccatcacc cctgcaccct atgtcaccat caaactgggg ggcagatcag
1967
tgagaggaca cttgatggaa agcaatacac tttaagactg agcacagttt cgtgctcagc
2027
tctgtctggt gctgtgagct agagaagctc accacataca tataaaaatc agaggctcat
2087
gtccctgtgg ttagacccta ctcgcggcgg tgtactccac cacagcagca ccgcaccgct
2147
ggaagtacag tgctgtcttc aacaggtgtg aaagaacctg agctgagggt gacagtgccc
2207
aggggaaccc tgcttgcagt ctattgcatt tacataccgc atttcagggc acattagcat
2267
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ccactcctat ggtagcacac tgttgacaat aggacaaggg ataggggttg actatccctt
2327
atccaaaatg cttgggacta gaagagtttt ggattttaga gtcttttcag gcataggtat
2387
atttgagtat atataaaatg agatatcttg gggatggggc ccaagtataa acatgaagtt
2447
catttatatt tcataatacc gtatagacac tgcttgaagt gtagttttat acagtgtttt
2507
aaataacgtt gtatgcatga aagacgtttt tacagcatga acctgtctac tcatgccagc
2567
actcaaaaac cttggggttt tggagcagtt tggatcttgg gttttctgtt aagagatggt
2627
tagcttatac ctaaaaccat aatggcaaac aggctgcagg accagactgg atcctcagcc
2687
ctgaagtgtg cccttccagc caggtcatac cctgtggagg tgagcgggat caggttttgt
2747
ggtgctaaga gaggagttgg aggtagattt tggaggatct gagggc
2793
<210> 2
<211> 167
<212> PRT
<213> Murine
<400> 2
Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp Ser Tyr Leu
-1 1 5 10 15
Ser Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys
20 25 30
Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr
35 40 45
Gln Ser Val Ser Ala Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro
50 55 60
Gly Leu His Pro Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala
65 70 75
Val Tyr Gln Gln Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln
80 85 90 95
Ile Ala Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala
100 105 110
Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro
115 120 125
Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val
130 135 140
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Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln
145 150 155
Leu Asp Val Ser Pro Glu Cys
160 165
<210> 3
<211> 700
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (46)..(546)
<220>
<221> sig_peptide
<222> (46) . . (48)
<220>
<221> mat peptide
<222> (49)..(546)
<220>
<223> Human ob (leptin) where N represents adenine or
guanine or cytosine or thymine
<400> 3
nnngnngttg caaggcccaa gaagcccann ntcctgggaa ggaaa atg cat tgg gga 57
Met His Trp Gly
-1 1
accctgtgcggattc ttgtggctttgg ccctatcttttc tatgtccaa 105
ThrLeuCysGlyPhe LeuTrpLeuTrp ProTyrLeuPhe TyrValGln
5 10 15
getgtgcccatccaa aaagtccaagat gacaccaaaacc ctcatcaag 153
AlaValProIleGln LysValGlnAsp AspThrLysThr LeuIleLys
20 25 30 35
acaattgtcaccagg atcaatgacatt tcacacacgcag tcagtctcc 201
ThrIleValThrArg IleAsnAspIle SerHisThrGln SerValSer
40 45 50
tccaaacagaaagtc accggtttggac ttcattcctggg ctccacccc 249
SerLysGlnLysVal ThrGlyLeuAsp PheIleProGly LeuHisPro
55 60 65
atcctgaccttatcc aagatggaccag acactggcagtc taccaacag 297
IleLeuThrLeuSer LysMetAspGln ThrLeuAlaVal TyrGlnGln
70 75 80
atcctcaccagtatg ccttccagaaac gtgatccaaata tccaacgac 345
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Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp
85 90 95
ctg gag aac ctc cgg gat ctt ctt cac gtg ctg gcc ttc tct aag agc 393
Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
100 105 110 115
tgc cac ttg ccc tgg gcc agt ggc ctg gag acc ttg gac agc ctg ggg 441
Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly
120 125 130
ggt gtc ctg gaa get tca ggc tac tcc aca gag gtg gtg gcc ctg agc 489
Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser
135 140 145
agg ctg cag ggg tct ctg cag gac atg ctg tgg cag ctg gac ctc agc 537
Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser
150 155 160
cct ggg tgc tgaggccttg aaggtcactc ttcctgcaag gactnacgtt 586
Pro Gly Cys
165
aagggaagga actctggttt ccaggtatct ccaggattga agagcattgc atggacaccc 646
cttatccagg actctgtcaa tttccctgac tcctctaagc cactcttcca aagg 700
<210> 4
<211> 167
<212> PRT
<213> Homo Sapiens
<400> 4
Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu
-1 1 5 10 15
Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys
20 25 30
Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr
35 40 45
Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro
50 55 60
Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala
65 70 75
Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln
80 85 90 95
Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala
100 105 110
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Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu
115 120 125
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
130 135 140
Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln
145 150 155
Leu Asp Leu Ser Pro Gly Cys
160 165
<210> 5
<211> 146
<212> PRT
<213> Mus musculus
<220>
<223> Mature mouse ob (leptin)
<400> 5
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
1 5 10 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ala
20 25 30
Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45
Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Val
50 55 60
Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln Ile Ala Asn Asp Leu
65 70 75 80
Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys
85 90 95
Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu Asp Gly
100 105 110
Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val Ser Pro
130 135 140
Glu Cys
145
<210> 6
<211> 146
<212> PRT
<213> Homo sapiens
<220>
<223> Mature human ob (leptin)
<400> 6
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Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
1 5 10 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
20 25 30
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
35 40 45
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
65 70 75 80
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
85 90 95
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
100 105 110
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
