WO2001013935A2

WO2001013935A2 - Modulation of the blood-brain barrier transporter for leptin

Info

Publication number: WO2001013935A2
Application number: PCT/US2000/023110
Authority: WO
Inventors: William A. Banks
Original assignee: The Administrators Of The Tulane Educational Fund
Priority date: 1999-08-23
Filing date: 2000-08-23
Publication date: 2001-03-01
Also published as: EP1250131A2; WO2001013935A3; AU7066400A; CA2382666A1; MXPA02001882A

Abstract

Agents selected from the group comprising adrenergic agonists, adrenergic antagonists, neurotransmitters, cytokines, amino acids, opiate peptides, purinergic agonists, glutaminergic agonists, more particularly consisting of epinephrine, isoproterenol, arterenol, cirazoline, yohimbine, phentolamine, prasozin, benoxathian, TNF-a, tyrosine, adenosine and glutamate are used alone or in combination with leptins for modulating the transport of leptin across the blood-brain barrier, modulating body weight in mammal and for modulating appetite in a mammal.

Description

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., J. 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 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 barrier 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):305-31 1 (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. The mechanism of transport of proteins and peptides such as leptin across the blood-brain bamer is poorly understood Some proteins/peptides cross this barrier by diffusing directly through the endothehal/ependymal membranes according to their lipophihcity and/or molecular weight with smaller lipophihc molecules passing more freely [Banks & Kastin, Psxchoneuroendocrmology, 10 385-399

(1985)] Others are transported by saturable, earner-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 bamer is through receptor-mediated permeabihzation which is enabled by the administration of molecules such as bradykinin, leukotπenes, histamme, and 5-hydroxytryptamme [Unterberg, A , J Cereb Blood Flow Metab , 4 574-585 (1984)] Similarly, molecules such as leucme encephahn, -adrenergics, arachidomc acid, aluminum, phorbomyπ state esters, and α-thrombm increase blood- brain barrier permeability while angiotensm II and β-adrenergics reduce the permeability [Gπeg, N , Physiology and Pharmacology of the Blood-Brain Barrier Handbook of Experimental Pharmacology, 103 487-523 Spπnger-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- bram barrier

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 alteπng the transport of leptin across the blood-brain barrier According to the invention, leptin transport across the blood- brain bamer may be increased, thereby resulting in a reduction in body weight, and/or a decrease in appetite Conversely, leptin transport across the blood-brain bamer 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-mduced cachexia)

The compositions may act on either side of the blood-bram barrier (or other blood- tissue barriers) to result in altered transport of leptin, although preferred compositions and methods act on the blood side of the barrier

A preferred method of the invention compπses administering to a subject in need thereof a composition which compπses an adrenergic agonist in an amount effective to increase the transport of leptin or leptin vaπants, analogs, fragments, consensus leptin, or deπvatives (including but not limited to a fusion protein) or chemically modified deπvatives of leptin across the blood-bram bamer A fusion protein refers to a protein compπsing a leptin polypeptide and a different 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 deπvatives thereof or fusion protein) across the blood- brain bamer Conversely, in another embodiment of the invention, an adrenergic antagonist may be used to inhibit leptin transport across the blood-bram bamer 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 puπnergic and glutammergic agonists may also be used to modulate leptin transport into the brain An exemplary puπnergic agonist compπses adenosine while an exemplary glutammergic agonist compπses glutamate

Routes of administration of the compositions useful in the practice of the invention include but are not limited to intravenous, lntraarteπal, intrapeπtoneal, intramuscular, mtradermal, topical, intraocular, subcutaneous, intranasal, oral, intracisternal, intracerebroventπcular, intrathecal, topical, mtradermal, or pulmonary Conversely, in another preferred embodiment, an adrenergic antagonist may be used to inhibit leptin transport across the blood-brain barπer

In one embodiment of the present invention, the composition compπses one or more compounds selected from the group consisting of adrenergic agonists such as, but not limited to, epinephπne, isoproterenol, arterenol, cirazohne, phenylethylamine, epinephπne, norepinephπne, dopamme, nordefπn, protokylol, metaproterenol, metarammol, phenylehpπne. tyramine, hydroxyamphetamine, nyhdπn, isoxsupπne, methoxyphenamine, methoxamine, amphetamine, methamphetamine, ephedπne, phenylpropanolamme, mephentermme, chlorphentermine, tuaminoheptane, cyclopentamine, propylhexedπne, and analogs and deπvatives or metabolites thereof and optionally, a pharmaceutically acceptable earner, excipient or diluent Exemplary adrenergic antagonists includes, but are not limited to, phentolamme, prazosm, benoxathian, phenotybenzamme, and related laloallyl-aminos In another embodiment, the compositions of the invention compnse punnergic and glutammergic agonists or combinations thereof and their use in the methods of the invention Adenosine activates the adenosine, or punnergic 1 (PI) receptor PI 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 hgand for glutamate (glutammergic) receptors Glutamate receptors include lonotropic 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 barπer and which are useful in the practice of the present invention include free fatty acids, sugars such as glucose, cytokmes, drugs such as amphetamines, calcium channel blockers, monoamines, amino acids, hormones including steroid hormones, dietary supplements, ketones. starches, micronutπents, hpoproteins, prostaglandins, prostacychns, peptides, proteins, regulators of nitnc oxide production, NMDA and GABA agonists and antagonists, vitamins, minerals, and melatonm, and their precursors and metabolites Preferred cytokines useful in the practice of the present invention include, but are not limited to, interleukin lα, mterleukm l β, interleukin 1 receptor antagonist, interleukin 2, mterleukm 6, mterleukm 12, macrophage colony stimulating factor, macrophage inflammatory peptides such as MIP-l α, MlP-l β, and tumor necrosis factor α (TNFα) Other compounds used in the practice of the present invention include fenflunmine and related compounds

Other peptides and proteins useful in the practice of the present invention either alone or in combination with other compounds descnbed herein include, but are not limited to, adrenocorticotropm hormone (ACTH), amylm, atnal natnuretic peptide (ANP), bombesin, calcitonm, calcitonm gene related peptide

(CGRP), caerulem, cocaine and amphetamine regulated transcπpt peptide (CART), cholecystkinms (CCK), corticotropin releasing hormone (CRH), Cyclo-His-Pro, enterostatm, FMRF-amide, galanin, glucagon, glucagon-hke peptide (GLP), growth hormone, growth hormone releasing hormone (GHRH), gonadotropin hormone releasing hormone (GnRH or LHRH), insulin, msuhn-hke growth factors, macrophage migration inhibiting factor, melanocyte stimulating hormone (MSH), motilin, MSH-inhibitory peptide (MIF-1), nerve growth factor (NGF), neuromedms, neuropeptide Y (NPY), neurotensm, neurotrophms (NT-3, NT-4), opiate peptides (endorphins, enkephalms, endomorphins, dynoorphins, kyotorphin), orexm, oxytocin, pancreatic polypeptide, parathyroid hormone (PTH), pituitary adenylate cyclase activating polypeptides (PACAP), sauvagine, somatostatin, substance P, thyroid stimulating hormone (TSH), thyrotropm releasing hormone (TRH), tyrosine MIF-1, vasoactive intestinal polypeptide, and vasopressms

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 This invention is also directed to a method for treating obesity which compπses enhancing the transport of leptin, leptin variants, analogs, consensus leptins, fragments, or leptin derivatives thereof across the blood-brain barner 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 melhtus, including type I and type II diabetes and insulin-resistant pathologies which compnse enhancing the transport of leptin, leptin vanants, analogs, consensus leptins, fragments, or derivatives thereof across the blood-brain barner according to any of the preceding aspects or embodiments are provided

Also with the scope of the present invention are pharmaceutical compositions useful for modulating body weight, the composition compnsing leptin compπsmg the ammo acid sequence set out in SEQ ID NO 2 or 4, SEQ ID NO 5 and SEQ ID NO 6, consensus leptins, vanants, analogs, leptin fusion proteins, chemically modified deπvatives of leptin, and fragments thereof, and one or more agents selected from the group consisting of adrenergic agonists, adrenergic antagonists, neurotransmitters, peptide hormones, cytokines, ammo acids, opiate peptides, puπnergic agonists, puπnergic antagonists, glutammergic agonists and glutammergic 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 bamer

The invention also compnses the use of adrenergic agonists, adrenergic antagonists, neurotransmitters, peptide hormones, cytokines, ammo acids, opiate peptides, punnergic agonists or antagonists, glutammergic 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 compnse the use of any of the leptms 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 bram 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 bram via a specific saturable leptin transporter located at the blood brain barner Obesity can occur when the brain incorrectly senses less than the appropπate amount of leptin in the organism which thereby tπggers mechanisms to increase adiposity (e g , increasing feeding, decreasing metabolic rate) Adiposity then increases until the bram senses an appropπate level of leptin It, therefore, follows that increasing the efficiency of transporting leptin across the blood brain barπer 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 barner is associated with or contains withm 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 allestenc 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- bram 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 barπer and mateπals and methods for modulating appetite

More particularly, the present invention is directed to compositions including pharmaceutical compositions and methods for enhancing or inhibiting the transport of leptin (OB) polypeptides across the blood-brain bamer 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 melhtus (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 barπer to a site of action in the bram

For the purposes of this invention, leptin and OB are used interchangeably and refer to a polypeptide having as a mature form about 146 ammo acids

Any leptin molecule, including leptin vanants, analogs, fragments, consensus leptins, or denvatives, which have the ability to modulate weight, or to alter metabolism m a host mammal, is useful in the practice of the present invention. Preferred leptm proteins useful in the practice of the present invention may be native munne leptin set out as SEQ ID NO 2 which includes its signal sequence, or its mature form beginning at ammo acid 21 (as numbered in SEQ ID NO 2) of native leptin and set out as SEQ ED NO 5 or protein as set forth in Zhang et al (Nature, supra, herein incorporated by reference) or the native human OB protein (SEQ ID NO 4) or its mature sequence beginning ammo acids 21 through 166 set out as SEQ ID NO 6 (See Zhang et al , Nature supra, at page 428 ) Vanants 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 ammo acids with another while still maintaining a biological activity of leptin Natural vanants of either leptin which lack a glutamme residue at position 28 of the mature sequence or other natural vanants 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 compπses 1) an arginme m 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- >K¹\ L->I⁷⁴) The leptin molecules useful in the practice of the present invention may also optionally compnse a methionine at the N-terminus (-1 position) The munne leptin protein has significant homology to the human protein, particularly as a mature protein, and, further, particularly at the N-termmus One may prepare an analog of the recombmant human protein for use in the practice of the present invention by alteπng (such as substituting amino acid residues), in the recombmant human sequence, the ammo acids which diverge from the munne sequence For example, using a human protein having a lysine at residue 35 and an lsoleucme at residue 74 according to the numbenng of SEQ ED NO 6, wherein the first amino acid is valme, and the amino acid at position 146 is cysteine, one may substitute with another ammo 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, 1 11, 118, 136, 138, 142, and 145 One may select the amino acid at the corresponding position of the munne protein, (SEQ ED NO 6), or another ammo acid

One may further prepare "consensus" molecules (consensus leptm or consensus OB) based on the rat OB protein sequence [Murakami et al , Biochem Biophys Res Comm 209 944-952 (1995) herein mcoφorated by reference] Rat OB protein differs from human OB protein at the following positions (using the numbenng of SEQ ED NO 6) 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108, HI, H8, 136, 138, and 145 One may substitute with another amino acid one or more of the ammo acids at these divergent positions The positions underlined and in bold pπnt are those in which the munne leptm 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 ammo acid from the corresponding rat OB protein, or another amino acid The positions from both mature rat and mature munne 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 ED NO 6 having one or more of the above amino acids deleted or replaced with another amino acid such as the ammo acid found in the corresponding rat or munne sequence may also be effective 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(1), 66(1), 67(N), 68(L), 89(L), 100(L), 108(E), 1 12(D), and 1 18(L) Since the recombmant 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 ammo acids replaced with another amino acid, such as the amino acids m parentheses, may be effective It should be noted that certain rhesus divergent ammo acids are also those found in the above munne species (positions 35, 68, 89, 108, and 1 18) Thus, one may prepare a munne/rat/rhesus/human consensus molecule (using the numbenng of SEQ ED NO 6 having one or more of the amino acids at positions replaced by another ammo 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, 1 1 1, 1 12, 1 18, 136, 138, 142, and 145 The positions underlined and in bold pnnt 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 leptm protein molecules (using the number of SEQ ED 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) 1 12-146

(e) amino acids 1-99 and (connected to) 1 12-146 having one or more of amino acids 100-1 1 1 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 munne OB protein) from human OB protein Furthermore, any alterations may be in the form of altered ammo acids, such as peptidomimetics or D-amino acids Further, leptin molecules having 83% or more amino acid identity with leptins having the amino acid sequence set out in SEQ ID NOs 2, 4 5 or 6 may also be used in the practice of the invention

Any of the foregoing leptm molecules may optionally have an N- termmal methionme 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 incoφorated 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 denvatized by the attachment of one or more chemical moieties to the protein moiety The chemically modified deπvatives may be further formulated for lntraartenal, intrapentoneal, intramuscular, subcutaneous, intravenous, oral, nasal, pulmonary, topical, ocular, intracisternal, intrathecal, transdermal, lntracerebroventncular, 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 lmmunogemcity 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 descnbing protein modification and fusion proteins is Francis, Focus on Growth Factors 3 4-10 (May 1992) (published by Mediscnpt, Mountview Court , Fπern Barnet Lane, London N20, OLD, UK)

The chemical moieties suitable for denvatization 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/prote conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations For the present proteins and peptides, the effectiveness of the deπvatization may be ascertained by admimsteπng the deπvative, in the desired form (1 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 descnbed 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 pyrohdone, poly-1, 3- dioxolane, poly-1, 3, 6-tπoxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random or non-random copolymers), and dextran or poly (n-vmyl pyrohdone) polyethylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinyl alcohol Polyethylene glycol propionaldenhyde may have advantages in manufactunng due to its stability in water Fusion proteins may be prepared by attaching polyaminoacids to the

OB protein (or analog) moiety For example, the polyammoacid may be a earner protein which serves to increase the circulatory half-life of the protein For the present therapeutic or cosmetic puφoses, such polyammoacid should be those which do not create neutralizing antibody response, or other adverse response Such polyammoacid 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_c") or other polyaminoacids As indicated below, the location of attachment of the polyammo 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- termmus with the N-terminus However, OB may be fused at its N-termmus with the C-termmus 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 CHI domain of the heavy chain 01 the corresponding region of the light chain The exact site at which the fusion is made is not cntical The fusion proteins may compnse 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 manufactunng 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 antigemcity 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- denvatize, or may provide for a di-, tπ-, tetra- or some combination of deπvatization, 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 denvatization (e g , mono, di-, tπ-, 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 antigemc 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 mcoφorated by reference (coupling PEG to G-CSF), see also Malik Exp Hematol 20 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chlonde) For example, polyethylene glycol may be covalently bound through ammo 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 ammo acid residues having a free amino group may include lysine residues and 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 puφoses is attachment at an ammo 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 vaπety 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-termmally 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 punfication of the N-terminally pegylated mateπal 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 pnmary ammo groups (lysine versus the N-termmal) available for denvatization m a particular protein Under the appropπate reaction conditions, substantially selective denvatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved For example, one may selectively N-termmally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pK_a differences between the e-ammo group of the lysme residues and that of the -amino group of the N-terminal residue of the protein By such selective denvatization, attachment of a water soluble polymer to a protein is controlled the conjugation with the polymer takes place predominantly at the N- termmus 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 descπbed above, and should have a single reactive aldehyde for coupling to the protein Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used An N-terminally monopegylated derivative is preferred for ease in production of a therapeutic, N-termmal pegylation ensures a homogenous product as charactenzation of the product is simplified relative to di-, tπ- 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 manufactunng

As descnbed in the following examples, the administration of compositions which interact with an adrenoreceptor (preferably adrenergic agonists) either pnor to or concurrently with the administration of leptin significantly increases the amount of leptin which crosses the blood-brain barner into the brain The compositions and methods of the invention are also useful for increasing the transport of endogenous leptin across the blood-bram barner These results are illustrated by the following Examples in which radiolabelled leptm was administered then measured m mice who were given compositions which interact with an adrenoreceptor Compositions containing epinephπne (which reacts with an adrenoreceptor) were the most effective in enhancing leptin transport Other compositions including those containing ammo acids or hormones were tested as well and in some cases were shown to be effective in enhancing leptin transport across the blood-bram bamer

The invention is descπbed 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 descnbes the effects of epinephπne on leptm transport across the blood-brain barner

Example 2 descnbes the effect of vanous dosages of epinephπne on the transport of leptin across the blood-bram barπer In Example 3, the effect of epmephrme on the integnty of the blood- brain barrier was examined

In Example 4, the effect of the amino acids tyrosine and phenylalanme on transport of leptin across the blood-bram barner was studied

In Example 5. the effect of arginme, phenylalanme, tryptophan, and tyrosine on the transport of leptin across the blood-brain barner was studied In Example 6, the effects of neurotransmitters including dopamine, histamine, serotonin, and epmephnne on leptin transport are described.

Example 7 descnbes the effect of co-admimstration of the adrenoreceptor agonists/antagonists cirazohne hydrochlonde, UK14304, albuterol, CGP- 12177 A, and benoxathian hydrochlonde 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, clomdine, arterenol, and phenylephnne 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 barner was tested.

Example 10 descnbes the effect of tumor necrosis factor on leptm transport across the blood-bram barner. Example 1 1 descnbes the effect of punnergic and glutammergic agonists on the transport of leptm across the blood-brain barner.

EXAMPLE 1

Effects of Administration of Epmephnne on Transport of Leptin across the Blood-Bram Bamer in Mice In this Example, the effect of administenng epinephπne on the transport of leptin across the blood-bram barπer in mice was studied.

En these expenments, 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 epmephnne (33 μg/200 μl) 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 (nun.), 30 mm., 45 mm , 1 hour (h), and 2 h post epmephnne injection, radiolabelled leptin (^l25I, 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 epmephnne, only lactated Ringer's solution with 1% bovme serum albumin and were injected with ^l2T- leptin only after the time interval of 10 minutes All mice were decapitated and their blood collected after 10 minutes following the leptin injection The bram (except pituitary and pineals) was removed and counted m a gamma counter (Micromedic 4/200, Horsham, PA) for 3 minutes Blood was collected from a cut in the nght carotid artery, centπfuged at 2000 g for 10 mm at 4°C, then 0 1 ml was counted in a gamma counter Bram blood ratios were expressed as counts algebraically to μl/g of brain over counts/min /μl of arteπal blood

Table 1 Administration of Epmephnne Followed by Leptin

The results of this expenment indicate that the administration of epmephnne pπor to the administration of leptm enhances the uptake of leptm by the bram in mice More specifically, in the control mice who received no epmephnne, 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/mm /μl of arteπal blood as compared to the amount of leptin in the mice who received epmephnne prior to leptin administration was 23 69+3 90 counts/min /g of brain over counts/min /μl of arteπal blood This represents an enhancement in leptin uptake by the brain of approximately 51% The other time points of 30 mm , 45 mm , 1 h, and 2 h illustrate that epmephnne still exerts its positive effects on leptin uptake by the brain even after a time interval of 2 h has passed EXAMPLE 2

Effects of Administration of Epinephnne 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.

As in Example 1, mice were anaesthetized with ethyl carbamate. The mice were then given an i.v. injection of a solution containing radiolabelled leptin (ⁱ²T, 2.1 x 10⁶ cpm) in lactated Ringer's solution with 1% bovine serum albumin and various amounts of epinephrine (133.33 μg, 400 nM; 66.6 μg, 200 nM; 33.3 μg, 100 nM; 13.3 μg, 40 nM; 0.667 μg, 2 nM) in 200 μl. 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

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. EXAMPLE 3

Effects of Administration of Epmephnne on the Integrity of the Blood-Brain Barrier in Mice

In this Example, the effects of the administration of epmephnne on the integrity of the blood-brain barπer in mice was evaluated Radiolabelled albumin is the traditional standard to be administered and monitored in order to test the mtegnty of the blood-brain barrier (Davson H (1967) Physiology of the Cerebrospinal Fluid, pp 82-103, J & A Churchill, London)

As descπbed above, mice were anaesthetized with ethyl carbamate then given an I v injection of either a solution containing radiolabelled leptin (¹²⁵I, 1 54 x 10⁶ cpm) and albumin ("Tc, 3 4 x 10⁶ cpm) (labeled solution) m lactated Ringer's solution with 1% bovme serum albumin in 200 μl or the labeled solution plus epinephπne (33 μg) All mice were decapitated with their blood and testis collected after 10 minutes following the leptin injection The bram (except pituitary and pmeals) 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 πght carotid artery, centπfuged at 2000 g for 10 min. at 4°C, then 50 μl was counted in a gamma counter. Bram/blood and testis/blood ratios were expressed as counts/min./g of brain or testis over counts/min./μl of arteπal blood.

Table 3 Co-administration of Epmephnne with Leptin and Albumin

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 barrier, as the amount of "Tc- 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 barrier, 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. 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 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 human leptin (^l25L 1.58 x 10⁶ cpm) in lactated Ringer's solution with 1% bovine serum albumin in 200 μl or the labeled solution plus one of the following amino acids (tyrosine or phenylalanme, 10 μg). 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

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 Leptin Across the Blood-Brain Barrier in Mice

En 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 (^,35I, 1.68 x 10⁶ cpm) in lactated Ringer's solution with 1% bovine serum albumin in 200 μl or the solution plus one of the following amino acids (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

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 barrier.

EXAMPLE 6

Effects of Administration of Neurotransmitters on Transport of Leptin Across the Blood-Brain Barrier 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 (icv) injection of a solution containing a neurotransmitter such as: acetylcholine, 98 μg; dopamine, 103 μg; epinephrine, 55 μg; histamine, 1 17 μg; or serotonin, 130 μg. Ten minutes later the mice were given an i.v. injection containing radiolabelled leptin (^12:>I, 1.77 x 10⁶ cpm) in lactated Ringer's solution with 1 % bovine serum albumin in 100 μl. 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

The results of this expeπment indicate that the icv administration of the neurotransmitters acetylcholme, dopamine, histamme, and serotonin with leptin had no effect on the uptake of leptm by the bram The administration of epmephnne by the icv route pnor to leptin also did not enhance the uptake of leptin by the bram This shows that the site at which epmephnne acts to modify leptin transport is on the blood side of the blood-brain barπer In another seπes of studies, neurotransmitters were injected intravenously with 200 nmol/mouse of either acetylcholme, dopamme, epmephnne, histamme, or serotonin The results of this study indicate that only epmephnne was capable of enhancing transport of leptm across the blood-brain barner

EXAMPLE 7 Effects of Administration of Adrenoreceptor Agonists on

Transport of Leptin Across the Blood-Bram Barrier in Mice

In this Example, the effect of certain adrenoreceptor agonists 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 labeled solution containing radiolabelled leptin (^l25I, 1 98 x 10⁶ cpm) in lactated Ringer's solution with 1% bovine serum albumin in 100 μl or the labeled solution plus one of the following agonists isoproterenol, 25 30 μg, clomdine, 18 66 μg, epinephrine, 14 26 μg, L- phenylephnne, 14 26 μg, or arterenol, 22 35 μg) Blood and brain samples were collected as descπbed above at 10 min post leptin injection

Table 7 Co-administration of Adrenoreceptor Agonists with Leptin

The results of this expenment indicate that the co-admmistration of the adrenoreceptor agonists, isoproterenol and arterenol with leptin enhanced the uptake of leptin by the bram However, clomdme and L-phenylephπne had no effect on leptin transport

EXAMPLE 8

Effects of Administration of Adrenergic Antagonists on Transport of Leptin Across the Blood-Brain Barner in Mice

In this Example, the effect of the co-administration of epmephnne with adrenoreceptor antagonists on transport of leptin across the blood-brain barπer was studied

As described above, mice were anaesthetized with ethyl carbamate The mice w ere then given an I v injection of either a labeled solution containing radiolabelled leptin (^l2l, 1 48 x 10⁶ cpm) in lactated Rmger's solution with 1% bovme serum albumin and epmephnne (3 33 μg) in 100 μl, or labeled leptin and epmephnne plus one of the following antagonists (phentolamine, 528 4 μg, D,L-propanolol, 4 41 μg, yohimbine, 547 3 μg, or prazosin, saturated solution of 587 8 μg) Blood and brain samples were collected as described above at 10 mm post leptin injection Table 8 Co-administration of Adrenoreceptor Antagonists and Epmephnne with Leptin

The results of this expenment indicate that the co-admmistration of the adrenoreceptor antagonists plus epmephnne either had no effect or reduced the uptake of leptin by the brain. Specifically, D,L-propanolol (a β 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 Adrenergic Agonists/Antagonists on Transport of Leptm Across the Blood-Brain Barπer 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 pnor Examples, mice were anaesthetized with ethyl carbamate. The mice were then given an 1 v. injection of either a solution containing radiolabelled leptm (^12,I, 1.2 x 10⁶ cpm) in lactated Ringer's solution with 1 % bovme serum albumin in 100 μl or the labeled solution plus one of the following agonists (cirazohne hydrochlonde, 25 μg; albuterol, 50 μg, UK 14304; epmephnne, 13.3 μg) or antagonists (benoxathian hydrochlonde, 250 μg + epmephnne, 13 3 μg; CGP-

12177A (250 μg) and epmephnne, 13.3 μg) Blood and brain samples were collected as descnbed in the previous Examples at 10 min post leptin injection. Table 9. Co-administration of α,, α₂ or β Adrenoreceptor Agonists/Antagonists with Leptin

The results of this experiment indicate that the co-administration of the α, agonist cirazoline with leptin enhanced the uptake of leptin by the brain. The data also show that the α, antagonist benoxathian blocked the enhancing effect of epinephrine.

EXAMPLE 10 Modulating Leptin Transport by Tumor Necrosis Factor a .TNF-α)

TNF-α (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. Three groups of mice w ere tested One group received labeled leptin (descπbed in Examples set out above) alone, a second group received labeled leptin plus 1 μg/mouse of mouse TNF, a third group received 1 μg /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 bram These data show that TNF and leptin do not share the same transporter (no competition or inhibition of leptm transport) nor does it acutely upregulate the leptin transporter (no enhancement of leptin transport) Chronic

This expeπment 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 companson 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 μl/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 μg/mouse of unlabeled leptin and 1 0 μg/mouse inhibited the entry rate by only 40% In normal mice, 0 3 μg inhibits entry by 50% and 1 μg/mouse inhibits entry by 95%

These data suggest that TNF receptor knockout mice have an altered transporter for leptin across the blood-brain barner Chronic TNF exposure, perhaps especially during development, is likely needed for the normal functioning of the leptm transporter This, therefore, represents another class of compounds (cytokines), in addition to the adrenergic agonist, ammo acids, and other compositions descπbed above that can modulate the transport of leptin across the BBB EXAMPLE 11 Modulation of Leptin Uptake b\ Adenosine and Glutamate

Studies were conducted to determine whether compounds which interact with punnergic receptors or with glutamate (glutammergic) receptors are capable of modulating the uptake of leptin into the brain

En 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 descnbed 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-Argmine is an essential ammo acid included below as a control After injection, brain and blood samples were obtained at 10 mm post leptm injection as descπbed The brain/serum ratios have been corrected for vascular space by subtracting 10 μl/g

Table 10 Co-admmistration of Adenosine, Arginine, or Glutamate with Leptin

The results of this expenment shown in Table 10 indicate that co-admimstration of the puπnergic agonist adenosine or the glutamate agonist significantly decreased the transport of leptin into the brain Co-admimstration of arginmes an essential amino acid had no effect on leptin transport

The results set out above show that punnergic agonists such as adenosine are useful in decreasing uptake of leptin into the brain and thus may act as 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 lonotropic receptors (e g AMP A, and N-methyl-D-aspartate receptors) and metabotropic receptors may also be useful in the same context as the punnergic agonists descnbed above.

While the present invention has been descπbed in terms of specific embodiments, it is understood that vaπations and modifications will occur to those skilled in the art. All of the foregoing references are hereby incoφorated by reference.

Claims

CLAIMSI CLAIM:

1. A method for modulating the transport of leptin across the blood-brain barrier of a mammal, the method comprising: administering to the mammal an effective amount of one or more compositions selected from the group consisting of adrenergic agonists, adrenergic antagonists, neurotransmitters, cytokines, amino acids, opiate peptides, purinergic agonists, glutaminergic agonists and metabolites thereof.

2. The method of claim 1 wherein said one or more compositions is administered to the mammal via a route of administration selected from the group consisting of intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, topical, intraocular, intracerebroventricular, intracisternal, intrathecal, intradermal, transdermal, nasal, oral and pulmonary.

3. The method of claim 1 further comprising co-administering to the mammal a leptin selected from the group of leptins comprising the amino acid sequence set out as SEQ ED NO: 2, SEQ ED NO: 4, SEQ ED NO: 5, SEQ ID NO: 6, consensus leptins, variants, analogs, leptin fusion proteins, chemically modified derivatives of leptin, and fragments thereof, said leptin optionally having an N- terminal methionine.

4. The method of claim 2 further comprising co-administering to the mammal a leptin selected from the group of leptins comprising the amino acid sequence set out as SEQ ID NO: 2, SEQ ED NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, consensus leptins, variants, analogs, leptin fusion proteins, chemically modified derivatives of leptin, and fragments thereof, said leptin optionally having an N- terminal methionine.

5 The method of claims 1 , 2, 3, or 4 wherein the one or more adrenergic agonists are selected from the group consisting of epinephπne, isoproterenol, arterenol and cirazoline

6 The method of claim 1 , 2, 3, or 4 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbine, phentolamine, prasozin, and benoxathian

7 The method of any one of claims 1 , 2, 3, or 4 wherein the cytokine is TNF-α

8 The method of claim 1, 2, 3, or 4 wherein the amino acid is

9 The method of claim 1, 2, 3, or 4 wherein the puπnergic agonist is adenosine

10 The method of claim 1, 2, 3, or 4 wherein the glutammergic agonist is glutamate

1 1 A method for modulating body weight in a mammal, the method comprising admimstenng to the mammal an effective amount of one or more compositions selected from the group consisting of adrenergic agonists, adrenergic antagonists, neurotransmitters, cytokines, amino acids, opiate peptides, puπnergic agonists, glutammergic agonist, and metabolites thereof

12 The method of claim 1 1 wherein said one or more compositions is administered to the mammal via a route of administration selected from the group consisting of intravenous, intraartenal, intramuscular, intraperitoneal, subcutaneous, topical, intraocular, intracerebroventncular, mtracisternal, intrathecal, mtradermal, topical transdermal, nasal, oral and pulmonary

13 The method of claim 1 1 further comprising co-admmisteπng to the mammal of a leptin selected from the group of leptins compnsing the amino acid sequence set out as ID No 2, SEQ ED No 4, SEQ ID No 5, SEQ ED No 6, consensus leptins, vanants, analogs, leptin fusion proteins, chemically modified denvatives of leptin, and fragments thereof, said leptin optionally having an N-terminal methionine

14 The method of claim 12 further compnsing co-admmisteπng to the mammal a leptin selected from the group of leptins compnsing the ammo acid sequence set out as SEQ ID NO 2, SEQ ED NO 4, SEQ ED NO 5, SEQ ED NO 6, consensus leptms, vanants, analogs, leptin fusion proteins, chemically modified denvatives of leptin, and fragments thereof, said leptin optionally having an N- termmal methionine

15 The method of claim 1 1 wherein modulating body weight is decreasing body weight

16 The method of claims 1 1, 12, 13, 14, or 15 wherein the one or more adrenergic agonists are selected from the group consisting of epinephπne, isoproterenol, arterenol, and cirazoline

17 The method of claims 1 1 , 12, 13, 14, or 15 wherein the amino acid is tyrosme

18 The method of claims 1 1 , 12, 13, or 14 wherein the cytokine is TNF-α

19 The method of claim 1 1 wherein modulating body weight is increasing body weight

20 The method of claims 1 1 , 12, or 19 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbine, phentolamine, prasozin, and benoxathian

21 The method of claim 11, 12, or 19 wherein the puπnergic agonist is adenosine

22 The method of claim 1 1, 12, or 19 wherein the glutammergic agonist is glutamate

23 A method for modulating appetite in a mammal, the method compnsing admimsteπng to the mammal an effective amount of one or more compositions selected from the group consisting of adrenergic agonists, adrenergic antagonists, neurotransmitters, cytokines, amino acids, opiate peptides, punnergic agonists, glutammergic agonists, and metabolites thereof

24 The method of claim 23 wherein said one or more compositions is administered to the mammal via a route of administration selected from the group consisting of intravenous, intraartenal, intramuscular, intrapentoneal, subcutaneous, topical, intraocular, intracerebroventπcular, intracisternal, intrathecal, mtradermal, topical, transdermal, subcutaneous, nasal, oral, and pulmonary

25 The method of claim 23 further comprising co-admmisteπng to the mammal a leptin selected from the group of leptms compnsing the amino acid sequence set out as SEQ ED NO 2, SEQ ED NO 4, SEQ ID NO 5, SEQ ED NO 6, consensus leptins, vanants. analogs, leptin fusion proteins, chemically modified deπvatives of leptin, and fragments thereof, said leptin optionally having an N- terminal methionine

26 The method of claim 24 further comprising the co- administeπng to the mammal a leptin selected from the group of leptins compnsing the amino acid sequence set out as SEQ ED NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, consensus leptms, vanants, analogs, leptin fusion proteins, chemically modified deπvatives of leptin, and fragments thereof, said leptins optionally having an

N-terminal methionine

27 The method of claims 23, 24, 25, or 26 wherein the one or more adrenergic agonists are selected from the group consisting of epinephπne, isoproterenol, arterenol, and cirazoline

28 The method of claims 23, 24, 25, or 26 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbine, phentolamine, prasozm, and benoxathian

29 The method of claims 23, 24, 25, or 26 wherein the cytokme is

TNF-α

30 The method of claims 23, 24, 25, or 26 wherein the amino acid is tyrosme

31 The method of claims 23, 24, 25, or 26 wherein said puπnergic agonist is adenosine

32 The method of claims 23, 24, 25, or 26 wherein the glutammergic agonist is glutamate

33 A pharmaceutical composition useful for modulating body weight, the composition compnsing a leptin selected from the group of leptms compnsing the amino acid sequence set out in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ED NO 6, consensus leptins, vanants, analogs, leptin fusion proteins, chemically modified deπvatives of leptin, and fragments thereof, said leptin optionally having an N-termmal methionine, in combination with an effective amount of an andrenergic agonist, adrenergic antagonist, neurotransmitter, cytokine, ammo acid, opiate peptide, puπnergic agonist, glutammergic agonist, and metabolites thereof

34 The pharmaceutical composition of claim 33 wherein the one or more adrenergic agonists are selected from the group consisting of epinephπne, isoproterenol, arterenol, and cirazoline

35 The pharmaceutical composition of claim 33 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbme, phentolamine, prasozin, and benoxathian

36 The pharmaceutical composition of claim 33 wherein the cytokine is TNF-α

37 The pharmaceutical composition of claim 33 wherein the amino acid is tyrosine

38 The pharmaceutical composition of claim 33 wherein said puπnergic agonist is adenosine

39 The pharmaceutical composition of claim 33 wherein the glutammergic agonist is glutamate

40 The use of one or more adrenergic agonists or metabolites thereof for the manufacture of a medicament for modulating the transport of leptin across the blood-brain barner

41 The use of claim 40 wherein the one or more adrenergic agonists are selected from the group consisting of epmephnne, isoproterenol, arterenol, and cirazoline

42 The use of one or more adrenergic antagonists or metabolites thereof for the manufacture of a medicament for modulating the transport of leptin across the blood-brain bamer.

43 The use of claim 42 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbme, phentolamine, prasozin, and benoxathian.

44. The use of one or more neurotransmitters or metabolites thereof for the manufacture of a medicament for modulating the transport of leptm across the blood-bram barner

45. The use of one or more peptide hormones for the manufacture of a medicament for modulating the transport of leptin across the blood-bram bamer.

46 The use of one or more cytokines for the manufacture of a medicament for modulating the transport of leptin across the blood-brain barner.

47. The use of claim 46 wherein the cytokine is TNF-α.

48 The use of one or more ammo acids for the manufacture of a medicament for modulating the transport of leptin across the blood-brain barner

49 The use of claim 48 wherein the amino acid is tyrosine

50 The use of one or more opiate peptides for the manufacture of a medicament for modulating the transport of leptin across the blood-brain barner 51 The use of one or more puπnergic agonists for the manufacture of a medicament for modulating the transport of leptin across the blood-brain barner

52 The use of claim 51 wherein said punnergic agonist is adenosine

53 The use of a glutammergic agonist for the manufacture of a medicament for modulating the transport of leptm across the blood-bram barner

54 The use of claim 53 wherein the glutammergic agonist is glutamate

55 The uses according to any one of claims 38 to 54 further compnsing the use of leptin for the manufacture of said medicament for modulating the transport of leptin across the blood-bram barπer

56 The use according to claim 55 wherein said leptin is selected from the group consisting of leptin compnsing the amino acid sequence set out as SEQ ID NO 2, SEQ ED NO 4, SEQ ED NO 5, SEQ ED NO 6, consensus leptins, vanants, analogs, leptin fusion proteins, chemically modified denvatives of leptin, and fragment thereof, said leptin optionally having an N-terminal methionine

57 The use of one or more adrenergic agonists or metabolites thereof for the manufacture of a medicament for modulating the body weight of a mammal

58 The use of claim 55 wherein the one or more adrenergic agonists are selected from the group consisting of epmephnne, isoproterenol, arterenol, and cirazoline 59 The use of one or more adrenergic antagonist or metabolites thereof for the manufacture of a medicament for modulating the body weight of a mammal

60 The use of claim 59 wherein the one or more adrenergic antagonists are selected from the group consisting of yohimbine, phentolamine, prasozin, and benoxthian

61 The use of one or more neurotransmitters or metabolites thereof for the manufacture of a medicament for modulating the body weight of a mammal.

62 The use of one or more peptide hormones for the manufacture of a medicament for modulating the body weight of a mammal

63 The use of one or more cytokines for the manufacture of a medicament for modulating the body weight of a mammal

64 The use of claim 63 wherein the cytokine is TNF.

65 The use of one or more ammo acids for the manufacture of a medicament for modulating the body weight of a mammal

66 The use of claim 65 wherein the ammo acid is tyrosine

67 The use of one or more opiate peptides for the manufacture of a medicament for modulating the body weight of a mammal

68 The use of one or more punnergic agonists for the manufacture of a medicament for modulating the body weight of a mammal 69 The use of claim 68 wherein said punnergic agonist is adenosine

70 The use of a glutammergic agonist for the manufacture of a medicament for modulating the body weight of a mammal

71 The use of claim 70 wherein the glutammergic agonist is glutamate

72 The uses according to any one of claims 57, 58, 65, or 66 further compnsing the use of leptin for the manufacture of said medicament for modulating the body weight of a mammal

73 The use according to claim 72 wherein said leptin is selected from the group consisting of leptm compnsing the amino acid sequence set out as SEQ ED NO 2, SEQ ED NO 4, SEQ ID NO 5, SEQ ED NO 6, consensus leptins, vanants, analogs, leptin fusion proteins, chemically modified denvatives of leptin, and fragments thereof, said leptin optionally having an N-terminal methionine

74 The use of any one of claims 57, 58, 65, 66, or 72 wherein modulating body weight is reducing body weight

75 The use according to claim 72 wherein modulating body weight is reducing body weight

76 The use of any one of claims 57, 59, 60, 68, 69, 70, or 71 wherein modulating body weight is increasing body weight