CA2562767A1 - Implantable pump for protein delivery for obesity control by drug infusion into the brain - Google Patents

Abstract

Methods and compositions are provided for suppressing appetite by surgically implanting a drug infusion pump into a site in a subject, and delivering a stable suspension of an appetite suppressing agent a region in a central nervous system of the subject. The appetite suppressing agent binds to a target receptor on a neural cell in the central nervous system and modifies the receptor function to suppress appetite.

Description

Y]VIPY.AN''Y'A:BLE I'CAVIP FOR PROTEIN DELIVERY FOR O$ESXTY CONTROL
BY DRUG YN'FCISION INTO THE BRAi.N

FI]r1:.D OF Ti-IE tNVENTION

[0001] The present invention relates to using implantable drug infusion pumps and methods for delivering appetite suppressing agents to the central nervous system.
BACICGROUND OF THE INVENTION

[0002] Recent studies have indicated that between a third and a half of all Amcricans are either overweight or obese (have a Body Mass Index (BMI) of greater than 25 kg/m2).
Inoreases in caloric intake coupled witll declines in exercise levels among the population have set the stage for a problem of epidemic proportions. The importance of addressing this problem and ultitnately treating obesity is emphasized by the fact that this disease is either the underlying cause, or a risk factor, for developing diseases such as type 11 diabetes, congestive hedri; failure, ostecarthritis and sleep apnea among others.

[0003] Currently, the primary treatment for obesity typically involves behavioral obange involving dietary restraints to reduce caloric intake coupled with aerobic and anaerobic exercise. Several dietary supplement drugs or other ingestib]e preparations are also used as appetite suppressors. In general, these techniques tend to produce only a temporary effeot.

[0004] Recently, melanocortin receptors have been found to play a major role in the regulation of energy balanoe and obesity in humans as well as other mammals.
In fact, weight loss has been found to resnlt from the pharmacological stimulation of inelnnocortin systeni activity. Tn rodents pharmacological stimulation of certain iuelanocortn receptors has lead to decreased food intake, increased energy expenditure and weight loss (Pierroz et a1.. Diabetes 51: 1337-1345 (2002)). In humans intranasal administration of Alpha-melanocyte stimulating hormone (alpha-MSH), a 13 amino acid peptide liormone, in non-obese men resulted in decreased body weight due to the loss of fat mass. (Fehm et al., J.
Clin. Endo. Metabol. 86: 1144-1148 (2001)).

[4005] To date, melanocortin receptor binding peptides such as alpha-MSH, have had lirnited use as pharmaceuticals due to their extremely short serum half life.
In addition, 'recent effortq to develop specific small molecule agonists of alpha-MSH have been slow;
none of these compounds liave advanced into the clinic.

[0006] Accordingly, a ireed exists for a more effective therapy for obesity and in particular more effective methods of delivering modulators of inelanocorin system activity to subjects are needed.

SUMMARY OF THE INVENTION

[0007] The invention provides systems, methods and compositions for suppressing appetite as a way to regulate body weight and obesity. In particular, the invention pertains to suppressing appetite by administering at least oue appetite suppressing agent that modulates the melanocortin system, an important pathway involved in weight gain and obesity.

[0008] The present invention involves suppressing appetite in a subject by implanting a drug infusion pump into a site in the subject. A stable suspension of an appetite suppressing agent carr then delivered to region in a central nervous system of the subject, e.g., a region of the brain. The appetite suppressi.ng agent can bind to a target receptor associated with appetite that is present on a neural cell, and modify the function of a receptor, to tliereby suppress appetite.

[0009] Yn aiwther aspect, the invention pertains to a method for ameliora.ting obesity in a subject by surgically implanting a drug infasion pump into a site in the subject. A stable suspension of a melanocortin, such as a melanocyte-s timufating hormone (MStI), can then be delivered to a site in the central nervous system of the subject. The malanocortin can bind to a melanocorhin receptor that is present on a neural cell, and modify the melanocortin receptor function to stippress appetite, to thereby amelioratc obesity.
DETAILED DESCRIPTION OF TffE XIriVENTTON

Tntroduction [0010] Certain exemplary embodiments of the invention will now be described to provide an overall understanding of the principles of the structure, function, manufactnre, and use of the methods and compositions disclosed herein. Those skilled in the art will understand that the methods flr-d compositions specifically described herein are non-limiting _2..

exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features ilhistrated or described in connection with one exemplary embodiment may be combined with tha features of otlier embodiments. Such modifications and variations are intended to be included within the scope of the present invention.

[0011] In particular, the invention pertains to suppressing appetite by administering an appetite suppressing agent that modulates the melanocortin system, an important pathway involved in weight gain. Melanocortins and melanocortin receptors play a major role in the regulation of overall energy balance and obesity in humans.

[0012] In one aspect of the invention, the appetite suppressing agent is delivered to a central nervous system target site (e.g., the intrathecal space, the brain) by an inmplantable drug infusion pump.

Definitions [0013] Various terms relating to the biological molecules of the present invention are used througliout the specification and claims.

[0014] "Obesity" means the abno.rmal accumulation of body fat, also referr d to as adipose tissue, above a medically relevaut threshold, such as a BMI exceeding 27 kg/mz.
The term is specifically meant to include subjects that have classically been categorized as both "overweight" and "obese."

[0015] "Isolated" means altered "by the hand ofrnan" fram the natural state.
If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the salne polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is employed herein.

[0016] "Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonueleotide, wh.ich may be unmodified RNA or DNA or modified RNA
or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- aud double-stranded regions, hybrid molecules comprising DNA and 'RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide"
xefers to triple-strauded regions comprising RNA or DNA or both RNA and DNA.

[0017] The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as iuosine. A variety of modifioations can been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymaticalIy or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligouucleotides.

[0018] "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptidc isosteres.
"Polypeptide" refers to both short chains, commonly referred to as peptides, oligopcptides or oligoiners, and to longer ohains, generally referred to as proteius.
Polypeptides may contain amino acids other than the gene-eneoded amino aeids.'"I'olypeptides"
include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification tecbniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs; as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varyiug degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from posttranslation natural processes or may be made by synthetic methods.

[00191 Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of various moiety groups, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or'lipid derivative, covalent attachment of plwsphotidylinositol, cross-linking, cyclization, disulfide bond fonnation, demethylation, tormation of covalent cross links, formation of cystine, formation of pyroglutamate, formylation, ganmma-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, aelenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins--Structure And Molecular Properties, 2nd Ed., T. F.
Creighton, W.
H. Freeman and Company, New York, 1993 and Wold, F,, "PosttranslatYonal Protein Modif.tcations: Perspectives and Prospects, pgs. 1- 12 in "Posttranslational Covalent Modification Of Proteins", B, C. Johnson, Ed., Academic Press, New'Y'ork, 1983; Seifter et ad., "Analysis far protein modificati.ons and nonprotein cofactors", MetlY.Enzymol 182:626-646 (1990) and REtttan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann NYAcad Sci 663:48-62 (1992) [0020] "Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polyn.ucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid suubstitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.

[0021 ] A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A
variant of a polynucleotide or polypeptide nray be a naturally occtirring such as an allelic variant, or it may be a variant that is not known to ocatlr naturally. Non-iiaturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct syntbesis. For instance, a conservative amino acid substitution may be made with respect to the amino acid sequence encoding the polypeptide.

C0022;1 A"eonservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced with another an-dno acid residue having a similar side chain. Fant.ilies of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysiuie, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side clkains (e.g., glycine, asparagine, glutarnine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, nmethionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, plienylalanine, tryptophnu, histidine).

[0023] The term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein (i.e. the structure, stability characteristics, subatrate specificity and/or biological activity of the protein). With particular reference to nucleic acid sequences, the term "substmltially the same" is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerrte codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
With reference to amino acid sequenoes, the term "substantially the same"
refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.

[0024] With respect to singl8-siranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generF-lly used in the art (sometimos termed "sttbstantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a sutistantially con,plemcntary sequence contained within a single-stranded DNA or RNA
molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-co.mplementary sequence.

[0025] With respect to oligonucleotide constructs, b-ut not limited thereto, the term "specifically hybridizing" refers to the association between two single-stranded nueleoti.de molecules of sufficiently complementary sequence to pennit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide construct with a subatantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridi2ation of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
[00261 The term "sulastantially pure" refers to a"preparation comprising at least 50-60%
by weight the compound of interest (e.g., nticleic acid, oligonucleotide, protein, etc.).
More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of inte.rest. Purity is measured by methods appropriate to the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0027] The term "expression cassette" refers to a nucleotide sequence that contains at least one coding sequence along with sequeuce elemeuts that direct the initiation and termination of transcription. An expression cassette may include additional secluences, including, but not timited to promoters, enhancers, sequences involved in post-transcriptional or post-translational processes, as well as appropriate terminator sequences.
[002$] A "coding sequence" or "coding region" refers to a nueleic acid molecute having sequence information necessary to produce a gene product, when the sequence is expressed.

[0029] The term "operably linked" or "operably inserted" means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the cocling sequence so as to enable expression of the coding saquence. This same definition is sometimes applied to the arrangement of other transeription control elements (e.g., enhancers and regulators) in an expression vector.

[0030] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terniinators, and the like, that provide for the expression of a coding sequence in a host cell.

[0031] The terms "promoter", "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polyinerase in a cell and initiating transcription of a downstream (3' d'er.ection) coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate trauscription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polyinerase.

[0032] The term "nucleic acid co:nstruct" or "DNA construct" is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and.
inserted into a vector for transfornvng a cell, in vitro or in vivo. This term may be used interchangeably with the term "transfomung DNA". Such a nucleic acid construct may contain a coding sequence for a gene produet of interest, along with a seleetable marker gene and/or a reporter gene.

[0033] A"heterologoits" regiou of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecuye in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the matnmalian genomic DNA in the genome of the source organism. In anoth.er example, a heterologou.s region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic codiug sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA
as defined herein.

[0034] The term "DNA constract", as defined above, is also used to refer to a heterologous region, particularly one construeted for use in transformation of a cell. A cell has been "transformed" or "transfected" or "transduced" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA
may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasnmid. With respect to eukaryotic cells, a stably transfortned cell is one in which the transforming DNA has become :in.tegrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.

[0035] "Delivery of a therapeutic element or agent" may be carried out through a variety ofineans, such as by using parenteral delivery methods such as intravenous a.r,cl subcutaneous injection, and the like. Such methods are known to those of skill in the art of drug delivery, and are fiuther described herein in the sections regarding phannaceutical preparations and. treatment [0036] By a "therapeutically effective amount" is meant an amount of the polynucleotide or protein of, or fragrneut thereof, tlzat when adminiatered to a subject is effective to bring about a desired effect (e.g., a decrease of body fat) within the subject.

Receptors, Peptides and Polynucleotides [0037] Melanocortin receptors are members of the G-protein coupled receptor class. To date, five melanocortin receptors have been identified, each having a unique tissue expression pattern. Examples of inelanocortin receptors iuclude, but are not liu-iited to, melanocortin- l receptor (MC1R), melanocottin-2 receptor (MC2R), melanocortin-receptor (MC3R), melanocortin-4 receptor (MC4R), and melanocor.tin-5 receptor (MCSR).

[0038] Details of melanocortin receptor genes and prateins are available, for example in, U.S. Pat. Nos. 5,703,220 and 5,710,265 to Yamada et ai.; U.S. Pat. No.
5,532,347 to Cone et al.; and PCT Publication WO 97/47316 and U.S. Pat. Nos. 5,908,609 and 5,932,779 to Lee et al_; which describe lqiown ruelanocortin receptors and the genes encoding such reaeptors. Each of these patents and PCT publication is incorporated herein by reference in its entirety.

[0039] Melanocortins am- cleavage products of pro-opiamelanocortin (POMC).
Examples of inelanocortins include, but are not limited to, adrenocorticotrophin (ACTH), alpha-melanocyte stimulating hormone (a-MSH), beta-melanocyte stimulating lwrmone (P-MSH), gamma-melanocyte stimulating hormone (y-MSH), and beta-endorphiii.

[0040] In one embodiment, the melanocortin is alplia-melanocyte stimulating lrormone (oc-MSH), a 13 amino acid peptide honnone that is an important component of the melanoeortin system encoding by the nucleic acid sequence TCCTACTCCA
TGGAGCACTT CCGCTGGGGC AAGCCGGTG (SEQ ID NO: 1) and having the amino acid sequence SYSMF-HFRWGKPV (SEQ ID NO: 2). Alpha-MSH is produced by the proteolytic processing of pro-opiomelanocortin released by the pituitary gland. Alpha-MSH binds with high affinity to the melanoeortin-4 receptor (MC4R), but also binds rnelanocortin-3 receptor (MC3R) and melanocortin-5 receptor (MC5R). MC4R is a G-coupled protein receptor found in the brain which, when stimulated by alpha-MSH
binding, causes dccreased food intake and increased fat oxidation. Ultimately, stimulation of melanocortin receptors such as MC4R results in weight loss.

Miinetibodies [00411 Also included within the scope of the invention are homologs of a-MSH, mimetica (peptide or non-peptide) of a-MSH, fusion proteins comprising a-MSI-I, conjugates of a-MSH, and any pharmaceutical salts of a-MSH. Bxemplary conjugates of a-MSH
peptides are those conjugated ta antibodies or antibody fragments, also referred to as mimetibodies, [0042] The present invention provides polypeptides having the properties of binding a melanocortin receptor and mimicking different isotypes of antibody immunoglobulin molecules such as ZgA, IgD, IgE) IgG, or IgM, an.d any subclass thereof, such as TgAi, IgA2, IgGi, IgG2, IgG3 or IgG4, or combinations thereof, herein after generally referred to as "mimetibodies." in some embodiments, the mimetibody polypeptides of the invention contain an alpha melanocyte stimulating hormone peptide (alpha-MSH) sequence and are designated melanocortin recoptor binding alph.a-MSH mimetibody. Such aipha-MSH
mimetibod.y polypeptides can bind rnelanocortin receptor 4 (MC4R) and, with equal and lower affinity, for MC3R and MC5R respectively. One result ofsuch melanocortin receptor binding can be the stimulation or inhibition of melanocortin receptor activity.
Stimulation can cause weight loss while inhibition may cause weight gain.

[0043] In oue embodiment the polypeptides of the invention have the generic formula (I):
(Mp-Lk-V'2-I-Ig-CH2-CH3)(q (I) wliere Mp is a melanocortin receptor binding molecule, Lk is a polypeptide or chemical linkage, V2 is a portion of a C-terminus of an immunoglobulin variable regioii, Hg is at least a portiam of an immunoglobulin variable hinge region, CH2 is an immunoglobulin heavy chain C112 constant region and CH3 is an. immunoglobulin heavy chA,in CH3 constant region and t is independently an integer of I to 10.

[0044] As used herein, "melanocorkin receptor binding molecule" means a molecule, which oan bind at least one melanocortin receptor such as MCI R, MC2R, MC3R, MC4R, and MCSR. A given peptide chain is a"melanocortin receptor" if it has at leawSt sS%
amino acid sequence identily to a known me]anocortin receptor sequence or the mature form of a known melanocortin receptor and can fanction as a 0-protein ooupled receptor.
Percent identity between two peptide chains can be determined by pairwise aligmnent using the default settings of the AlignX module of'Vector NTI v.9Ø0 (Invitrogen Corp., Carslbad, CA). An exemplary melanocorpn receptor binding molecule is the 13 amino acid alpba-MSH peptide having the nucleic acid sequence TCCTACTCCA
TGGAGCACTT CCGCTQ00GC AAGCCGGTG (SEQ ID NO: 1) and the amino acid sequence SYSMEHFRW'CrSCT'V (SEQ ID NO: 2). Other melanocortin receptor binding molecules include biologically "ve fragments of SEQ ID NO: 2 and other amino acid sequences that can bind a melanocortin receptor. Tlie term "biologically active fragment" as used herein, refers to a portion of an alpha-MSH peptide that can bind to a melanocortin receptor such as MC4R. The pcptidc sequence HFRW (SEQ. ID.
NO.
4) encoded by DNA sequence CA1'i'T'I'CGCT C;G (SEQ. ID. NO. 3) is an exemplary "biologically active fiagment" ofthe alpha-MSH peptide sequenee SYSMEHFRWGY.t'V
(SEQ II) NO: 2). The HFRW fragment has been incorporated into the structur.e of the synthetic melanocortin receptor activator molecule melanotan Il (MTII) (Fan et al., Natur'e 38S: 165-168 (1997)).

[0045] Incorporation of melanocortin receptor binding molecules in the mimei:ibody polypeptides of the invention provides for binding to melanocortin receptors with a wide rangc of affinities. The mimetibody polypeptides of the invention may bind a melanocortin receptor with a Kd less than or equal to about 10'7, 10's, 10,100, 10'" or 10'12 M.'Ihe range of obtained IC50 values for aMSH peptide, MTII peptide and aMSHMMB were 260-400 nM, 5-30 nM and 200-300 nM reapectively. The affinity of a -l l-mimetibody polypeptide for a melanocortin receptor can be determined experimentally using any stutable method. Such methods may utilize Biacore or ICinExA
instrumentation, ELISA or competitive binding assays. Mimetibody polypeptides binding specific melanocortin receptors with a desired affinity can be selected from libraries of'variants or fragments by techniques known to those skilled in the art.

[0046] An alpha-MSH peptide having the amino acid sequence shown in SEQ ID NO:

may be modified to obtain other melanocortin receptor binding molecules. Such modif"ications may comprise the incorporation of C-[X],-C motifs into the peptide to confonnationally constrain the peptide through the formation of disnlfide bonds. In a C-[X]r,-C motif, C is a cysteine residue, X is a amino acid residues and n is an integer necessary to achieve the required conformational constraint. In this instance n can be as little as I residue and as high as 50. Exemplary C-[Xlõ-C modified peptide sequences are shown below:

AGCTATAGCT GCGAACATTT TCGCTGGTGC AAACCGGTG (SEQ ID NO: 5) SER T'Y'R SER CYS GI.U HIS PHE ARG TRP CYS LYS PRO VAL (SEQ IDNO: 6) AGCTATTGCA TGGAACATTT'fCGCTC~G'rTGC AAACCGGTG (SEQ ID NO: 7) SER TYR CYS MET GLU HIS I'HE ARG TRP CYS LYS PRO VAL (SEQ ID NO: 8) AGCTGCAGCA TGGAACATTF TCGCTGGTGC AAACCGGTG (SEQ ID NO: 9) SER CYS SER MET GLU IIIS PHE ARG TRP CYS LYS PRO VAL (SEQ ID NO: 10) TGCTATAGCA TGC'xAACATTT TCGCTGGGGC TGCCCCrGTG (SEQ ID NO: 11) CYS TYR SER MET GLU HIS PHE ARG TRI' GLY CYS PRO VAL (SEQ ID NO: 12) [0047J The modification may also comprise the incorporation of a W1-[Xjõ-Wa motif into the peptide to conformationally constrain the peptide through the formation of a tryptophan zipper. In a Wa-[X]õ-Wa motif W is tryptopban residue, X is an anii.no acid, a is an integer usually 2, but can be from 1 to 10, and n is an integer necessary to achieve the required conformational constraint. In this instance n can be as little a 1 residue and as high as 50. Further, the sequence HFRW (SEQ ID NO: 4) present in the alpha-MSH
peptide may also be modified by substituting any residue in this sequence with any one of F. H, W and M; for example, HFRW (SEQ li) NO: 4) can be substituted to FHWM
(SEQ
XD NO: 14) coded by the n.ucloic acid sequence'Y'I"TCATTGGATG (SEQ ID NO: 13).
[0048] In the polypeptides of the invention, the linker portion (Lk) providas strnotural flexibility by allowing the mimetibody to have alternative orientations and binding properties. Exemplary linkers include non-peptide chemical linkages or one to 20 amino acids linked by peptide bonds, wherein the amino acida are selected from the 20 naturally occurring amino acids or other amino acids (e.g. D-amino acids, non-naturally occurring amino acids, or rare naturally occurring aniino acids), The linb:er porfion oan include a majority of amino acids that are sterically unhindered, such as glycine, alanine and serino and can include GS, poly GS or any combination or polymer thereof. Othor exemplary linkers within the scope of the invention may be longer than 20 residues and may include residues other than glycine, alanine and serine [0049] In the mimetibodies used in the invention. V2 is a portion of a caiboay terminal domain of an immunoglobulin variable region such as a heavy chain variable region; Iig is a portion of the hinge domain of an innnunoglobulin variable region such as a heavy cWn variabte region.; C112 is an immunoglobulin heavy chain C~12 constant region;
CH3 is an immunoglobulin heavy chain CI.13 constant region. It will be recognized by those slcilled in the art that the Cn3 region of the polypeptidos of the invention may have its C-terminal amino acid cleaved off when expressed in ceilain recombinant systems.

[0050] In the mimetibody polypeptides used in the invention Hg, CI12 or CH3 may be of the IgGI or IgG4 subclass. A sequence is oftha IgGI or IgG4 subclass if it is formed or developed from ay1 or 14 heavy chain respectively. A given peptide chain is a ry I or -y 4 heavy chain if it is at least 80% identical to a known y 1 or y 4 heavy chain sequence of a given species. Percent identity between two peptide chains can be detcrmined by pairwise alignment using the default settings of the AlignX module of Vector NTI
v.9Ø0 (Invitrogen Corp., Carlsbad, CA).

[0051] In the mimetibody polypeptides used in the invention Hg, CH2 or C113 may individually be of the IgGi or IgG4 subclass. The mimetibodies of the invention may also comprise combinations ofFig, CH2 or Cf13 elements from each subclass For example, Hg may be of the IgG4 subclass while CK2 and CH3 are of tl-m TgGl subclass.
Alternatively, Hg, Ca2 and CH3 may all of the 1gG4 or IgG i subclass. The IgGI and IgG4 subclasses differ in the number of cysteines in the hinge region. Most IgG type antibodies, such as IgGi, are homodixneric molecules made up of two identical heavy (fi) chains and two identical light (L) clains, typically abbreviated H2L2. Thus, these molecules are generally bivalent with respect to antigen binding due to the formation of inter,heavy chain disulfide bonds and both antigen bindiug (Fab) arms of the IgG molecule have identical binding specificity. IgG4 isotype heavy chains, in contrast, coutain a CpSC motif in tlieir hinge regions capable of forming either inter- or intra-heavy chain disulfide bonds, i.e., the two Cys residues in the CPSC motii'may disulfide bond with the corresponding Cys residues in the other H chain (inter) or the two Cys residues within a given CI'SC
motif may disulfde bond with each other (intrl). Sinco the HL pairs in those IgG4 molecules with intra-heavy chain bonds in the hinge region are not covalently associated with each other, they may dissociate into HL monomers that then reassociate with HL monomers derived from other IgG4 molecules forming bispecific, heterodinieric IgG4 molecules.
In vivo isomerase enzymes may facilitate this process. In a bispecific 'igGr aatilaody the two Fab "anns" of the antibody molecule differ in the epitopes that they bind.
Substituting Ser residues in the hinge region of IgG4 with Pro results in "IgG,-like beliavior," i.e., the molecules form stable disulfide bonds between heavy chains and therefore, air, not susceptible to HL exchange with other IgG; molecules.

[0052] The mim.etibody polypeptides of the invention may be made more IgGI-like, or IgG1-like by the modification of sites which are involved in disulfide bond formation and are present in the Hg-CH2-Cy3 portion of the mimetibody polypeptides. Such sites may be modified by removal, deletion, insertion or substitution with other amino acids. Typically, the cysteine residues present in disulfide bond associated motifs are removed or substitiil-ed. ltemovaJ of these sites may avoid covalent disulfide bonding with other cysteine-containing proteins present in the minietibody p.roduaing host cell or intra-heavy chain disulfide bonding in IgG4-based constructs while still allowing for noncovalent dimerization ofmimetibody Hb-Qj2--CH3 domains. Modification of such sites can permit the fortnation of bispecific mimetibody polypeptides with two differeut M
portions or prevent the formation of such bispecific speeies [0053] 71ie IgG, and IgG4 subclasses also differ in their ability to mediate complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotox'icity (ADCC).
CDC is the lysing of a target eell in the presence of complement. The complement activation pathway is initiated by the binding of the fa-st component of the complement system (Clq) to a molecule complexed with a cognate antigen. IgG, is a strong iuducer of the complement cascade and subsoquent CDC activity, while IgG4 has little complement-indneing activity. ADCC is a eell-mediated process in which nonspecific cytotoxic cells that express Fe receptors (FoRs) involved in ADCC (e.g., nataral killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The IgGI subclass binds witb high affinity to Fc receptora involved in ADCC and contributea to ADCC, while IgG4 binds only weakly to such receptors and has little ADCC inducing activity. The relative inability of igGq to activate effector functions auch as ADCC is desirable since delivery of the mimetibody polypeptide to cells without cell killing is possi'ble.

[0054] The CDC and ADCC activity of the mimetibody polypeptides of the invention may be modified by altering sites involved in CDC and ADCC present in the tig-portion of the mimetibody polypeptide. Such sites may be modified by removal, deletion, insertion or substitution with other amino acids: In the mimetibodies of the invention sites involved in CDC, such as the Clq binding site, are typically removed or otherwise modified to miniunize CDC activity. Additionally, Fc receptor binding sites involved in ADCC can also be similarly modified in the mimetibodies of the invention. In general, such modification will remove Fc reoeptor binding sites involved in ADCC
activity from the mimeh'bodies of the invention. The sabstitution of I.eu residues with Ala residues in the CH2 portion of the polypeptides of the invention is one example of a modification which can minimize ADCC activity in the polypeptides of the invention. The CH2 amino acid sequeoae.

[0055] Antibodies of both the IgG4 and IgGI isotypes contain FcRn salvage reeeptor binding sites. The FaRn salvage receptor helps maintain IgG antibody levels in the body by recycling or transporting IgG type antibodies across enodothelial cell layers such as those lining the inside of body cavities and blood vessels. The FeRn salavage receptor does this by binding IgGs that have enternd endothelial cells by nonspecific pinocytosis and preventing these IgG antibody molecules from being degraded in the lysosome of the cell. The result of such FcRn receptor activity is that the serum half-life of a molecule with an FcR.n binding site is extended relative to an otherwise identical molecule lacking such a site.

[0056] It is desirable that the Hg-CH2-CH3 portion of the mimetibodies of the invention contain a FcRn binding site at the junction of the Cu2 and Cu3 regions. It is expected that such FcRn sites will increase the serum half-life of the mimetibodies of the invention as well as impmve other pharmacokinetic properties relative to a raelanocortin receptor binding molecule, such as alpha-MSH alone. In the mimetibodies of the invention FcRn sites may be modified or added by removal, deletion, insertion or substitution of amino acids. Typically, such modifications are used to improve the binding of a given site to the FcRn.

[0057] Antibodies with different isotypes, such as IgGa and IgGi, may contain glycosylation sites. Glycosylation of these sites can alter the properties and activites of antibody moTecules. Antibody molecules may be N-glycosylated or 0-glycosylated. N-glycosylation of antibody amino acid msidue side chains containing nitrogen atoms (e.g., Asn) can modulate antibody Fc offector functions such as ADCC by eonferrinl; a cytolytie activity to N-glycosylated antibody molecules. This ADCC associatBd cytolytae aotivity causes the lysis of cells effected by such N-glycosylated antl'Uodies.
Alternatively, an ankibody molecule may be 0-glycoaylated by modification of amino acid residue side chains containing oxygen atoms (e.g., Ser or Thr). 0-glycosylation cau decrease the serurn half-life of an antibody moleoule through increased lectin mediated clearance of 0-glycosylated antibody molecules from the serum. Additionally, 0-glycosylation can cause undesirable increases in antibody heterogenoity due to differing extents of 0-glycosylation between various antibody nwlecules. Lastly, both O-glycosylarion and N-glycosylation can alter the structure dependent properties of antibody molecules such as binding affinity and immunogenicity.

[0058] Like the antibody rnolecules they mimic, the mimetibody polypeptides of the invention may also be post-translationatly modified by N-glycosylation and 0-glycosylation. In most instances, :it is desirable to limit the N-glycosylation of the mimetibodies of the invention to minin112e cytolytic activity. N-glycosylation can be limited by the removal or substitution of amino acid residues, such as Asn, which are typically N-glycosylated. It is also desirable to limit m imetibody 0-glycosylation to minimize lectin-mediated clearance, mimetibody heterogeneity and the alteration of stnicture dependent mimetibody properties such as binding affinity and immunogenicity.
One way to minimize 0-linked glycosylation in the mimetibodies of the invention is to substitute Ala residues for Thr residues in the V2 portion of the polypeptides of the invention.

[0059] The mononteric structure Mp-r,k-'V'2-Hg-CH2-Cit3 of the mimetibody polypeptides of the invention can be linked to "t" other monomers where t is an integer from I to 10.
Such linking can occur through non-covalent interactions or covalent linlcages such as a Cys-Cys disulfide bond. In this way multimeric structures such as dimers and higher order multimers of the polypoptides of the invention can be formed. It is expected that dimerization of the polypeptides of the invontion will increase the affinity of these polypeptides to melanocortin receptors such as MC4R. The tenn "multimers" as used herein means molecules that have quaternary structuro and are formed by the association of two or more subunits.

[0060] The polypeptides of tl-ie invention can optionally comprise at the aminci terminus, a amino terminal portion of an immunoglobulin variable region, designated V 1 as shown in p'ormula Ii:

(V 1-Mp-IJlc-V2..FIg-C}t2-Cfj3ht) (II) [0061 ] The polypeptides of the invention may also comprise secretory signals necessary to facilitate protein secretion or other signals necet:sary for protein trafficking in the cell.
T,hose skilled in the art will recognize the appropriate secretory signals.

[0062] In one embodiment the polypeptides of the invention comprise SEQ Il5 NO: 16 or 18. SEQ ID NO:18 represents a(VI-Mp-I1c V2-Hg-CI.j2-Cji3)(0 ) melanocortin receptor binding alpha,-MSIi polypetide of generic formula (I.i) which has a secretory signal fused to its amino terminus. SEQ ID NO: 16 represents a(1VIp-I.k-'V'2-Hg-Cb2-CH3)tt1 melanocortin receptor binding alpha-MSH polypetide of generic formula (I). No secretory signal is present in SEQ ID NO: 16. The relevant DNA and protein sequences are set forth in Tables 1A, 1B, 2A and 2B below:

TABLE 1A:

Melanocortin receptor binding alpha-MSH nilmetibody witliout secretory signal (D3YA) (SEQ ID NO:15).

tcctaatcca tggagcactt ccgctggggc aagocgstgg gatcoggtgg aggctccggt 60 aocttagtca ccgtctccto agagcecaaa tcttgtgaca aaactcacsc gtgcccaccg 120 tgccoagcao ctgaactcct ggggggaccg tcagtcttcc tcttcceccc aaaacccaag 180 gacaccctca tgaatctcccg gaoocctgag gtmoatgcg tggtggtgga cgtgagccac 240 gaagaccctg ag6tcaagu eeulcCuLqac gtggqcggcg tggaggtgea taatgccaag 300 acaaagccgc gggaggagca gtacaacago acgtaccgggtggtca cgtoctcACcgtc 360 ctgaaceagg actggctgaa tg eaaggag taeaagtgea antctcoaa eaaagccctc 420 ccagccccca tcgagaaaac catctccaaa gcoaaagggc agoceegaga acoaceggtg 4B0 tacaccctgc ccccatcccg ggatgagctg acoaagAacC amcagcct gacctgcctg 540 g0=aggct tctatcccag cgacatcgcc gtgg.~gtggg agagcaatgg gcagccggag 600 aacaaotaca agacc.lcgcc tcccgtgctg gactccgacg gctccttatt octctacage 660 aagctcaccg tggacaagag caggtggcag caggggeacg tcttctcatg ctccgtgatg 720 catgaggctc tgcacaaoo& etacaogoag aagagcctCt ccctgtctcc gggtaaa 777 -1$-TAR . 1B:

Melanacortin receptor binding alpfta-MSH mimetibody without secretory signal (PROTLIlY) (SEQ Yp NO: 16).

Ser Tyr Ser Met Glu Iiis Phe Arg Trp Gly I.ys Pro Val GIy Ser Gly Gly Gly Set Gly Thr Leu Val Thr Vxl Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lau Leu Gly Gly Pro Ser Val Phe I.eu Phe Pro Pro Lys Pro Lys Asp Thr l.eu Met lle Ser Arg Thr Pro Glu VaI Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 41u Val Lys Pho Asn Trp Tyr Val Asp Gly Val Glu Val rlis Asn Ala T.ys Thr Lys Pro Arg Olu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Va.l Ser Val Lcu Thr Val l.eu 14is Gln Aap Trp Lou Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lle Glu Lys '17v iie Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gin Val Tyr Thr Lcu Pro Pro Ser Arg Asp Glu Lou Thr T.ys Asn Gin Val Ser Lou Thr Cys Leu Val Lys Gly Phc Tyr Pro Ser Asp Jle Ala Val Glu 'ltp Olu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Scr Lys t.eu Thr Val Asp Lys Scr Arg Trp Gln Gin GlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala i.eu His Aen His Tyr Thr Gin i.ys Ser i.au Ser Leu Scr Pro Gly Lys T11.BLE 2A:

Melanocnrtin receptor binding alpba-MSH mimetTibody with sacretory sigtw and V1. (DNA) (S)EQ IA NO: 17).

atggcttggg tgtggacctt gctancetg atacggccg cccaaagtat acaacccag 60 etccagtcct actccat= ycactt0cgc tggggcaagc cggtgggatc cp,Etggaggc 120 tccggtacct tagTCaccgt otectcagag cccaaatctt gtCacaaaac tcacacgtgc 1$0 ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt cccaccaaaa 240 cccaaggaca ccctcatgat ctcccggacc cctgaggl:oa oatLticgtggt ggtggaegtg 300 agocacgaag accctgaggt caagttcaac tggtacgtgg aeggcEtgp pgtgcataat 360 gccaagacaa agccgeggga ggascagtac aacagcacgt ace(Wggt cagcgtcctc 420 accgtcctgc accaggactg gctgAatgga aaggagtaca agtgcaaggt ctccaacaaa 480 gccctcccag aceecatcga gaaaaccatotoeaaagcca aagggcpgcc ccgagaacea 540 caggtgtaca ccctgcccco atccagggat gagctgacca agaaccaggt caBcctgacc 600 tgcctggtea aaggcttata taccagagac atcgccgtgg agtgggagag caatgggcag 660 ccggagaaca acttxaagae eaegeetecc 8,tgctgsmct cegacggctc cttcttcete 720 taea$caage t'CaeCf'rtgg,a Ca4g3gCagg tggcagcagr UaaagtCCt Cteat$CteO 780 gtgat8catg aggctctgea caaceactac aCgcaWga gcctctccct gtctccgggt 840 aaa 843 T.aIBLE ZB-Melauoeortin receptor binding alpha-M91-I mimotibody with secretory signal and VI (PROTEIN) (SEQ rD NO:1$).

Mct Ala Trp Val Trp Thr Leu Leu Phe Lou Mct Ala Ala Ala Gln Ser Ile Gln Ala Gln Ilo Gin Ser Tyr Ser Met Glu His Phe Arr Trp Gly Lys Pro Val Gly Ser Gly Gly Gly Set G1y'I'hr Lcu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Tlu His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lcu Lou Gly Gly Pro Ser Val Phe Lou Phc Pro Pro Lys Pro Lys Asp Tln' Lea Met Ile Ser Arg Thr Pro Glu Vtil Thr Cys Val Val Val Asp Val Ser lils Glu Asp Pro Glu Val Lys Plte Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Qlu Gln Tyr Asn Ser Thr Tyr Arg VaI Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr I..ys Cys Lys Val Scr Asn Lys Ala Lou Pro Ala Pro Ilc Glu Lys Thr Ilo Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leti Pro Pro Ser Arg Asp Glu Lou 77'ir Lys Asn Gin Val Scr Lou Thr Cys L.,eu Val Lys Gly Phc Tyr Pro Ser Asp IIc Ala Val Glu T?p Glu Ser Asn Gly Gin Pro Glu Asn Azn Tyr Lys Thr Thr Pro Pro Val Lea Asp Scr Asp Gly Ser Phe Phe Lou Tyr 8cr Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gln Gly Asn V0.I
Pl,e Ser Cys Scr VaI Met His Glu Ala Lou His Asn His Tyr'fhr GIn Lys Ser Lou. Scr Lou Ser Pro Gly T.ys [0063] The invention also contemplates the use of polynucleotides comprising, complementary to or having significant identity with, a polynucleotide encoding at least one melanocortin receptor binding mimet'body. The invention also contemplates the use of a pharmaceutical composition comprising an effective amount of at least one mimetibody polypeptide and a pharmaceuticaIIy acceptable carrier or diluent.
The term "effective amount" generally refers to the quantity of mimetibody necessary for effective therapy, i.e., the partial or complete alleviation of the symptom or disorder for which treatment was sought. The composition can optionally eomprise at least one further compound, protein or composition useful for treating obesity and the other conditions described below. The phanmaceutically acceptable carrier or diluent in the compositions can be a solution, suspeusion, eni,ulsion, colloid or powder. Those slcilled in the art will recognize other pharmaceutically acceptable carriers and diluents.

[0064] The mirnetibodies employed in the present iuvention are fiuther described with reference to the following examples. Theso examples are merely to illustrate aspocts of the present invention and are not intended as limitations of this invention.

Exstmple 1 A1pha-MSII Mimetibodv and Exaression Vector Construction [0065] An alpha-MSH mimetiUody protein comprising a secretory signal sequence, an alpha-MSH peptide sequence, a linker sequence, VH ,sequenoe, a hinge sequenoe, a human.
igGl CH2 sequence and a Iiuman IgGi Cri3 sequence was designed (Tables 2A & 2B
and SEQ ID NO. 18) Analytyca.l data, e.g., mass spectroscopy, has confirmed that a mature polypeptide is generated (61,344.6 for G1/G1 form). Nucleic acid sequencas encoding this alpha-MSH mimetibody protein (SEQ ID NO: 17) were generated using standard molecular biology tecbniques. Nucleic acid sequences encoding the alpha-MSH
mimetibody sequence were subcloned into the p2389 expression vector to generate an alpha-MSH miinetibody expression vector.

Example 2 Alnha-MSR Mimetihodv Exnression [0066] The alpha-MSH mimetibody was transiently expressed in HEK293E cells.
Cells wara eulturod using standard conditions and transiendy transfected with the alpha-MSH
mirnetibody expressiou vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as direetad by the manufacturer. 24 h after transfection cells were transferred to a serum fiee media formulation and cultured for 5 days. The culture media was then removed and centrifuged to remove debris. Clarified media was incubated with Protein A-SapharoseTM
(1=Zix'rap rProtein A FF, Amersham Biosoiencies, Piscataway, NJ) and proteins were eluted from the Protein A-Sepbarose7m conjugate as directed by the manufacturer. '1 he cluted protein solution was then further purified via SuperoseTm 12 size excluaion chromatography (Superose 12 10/300 GL, Amersham 13-iosciencies, Piscataway, NJ) using standard methods. Column eluant was then subjcctod to SDS-pAGB and visualized by silver and Coomassie blue staining. Western blots were then prepared and the blots were probed with either an Fc specific primary antcbody or an alpha-MSH specific primary antibody. Together, the Westera Blot and SDS-PAGE staining results indicated that a purified alpha-MSH mimetibody, composed of two polypeptide chains, had been obtained from the transiently transfccted HEK293 cells.

E+ xSmple 3 Alnha-MSH Mimetibodv Binds MC4R

f0067] The alpha-MStX mimetibody binds to MC4R. and can compete with radiolabeled [Nle(4), D-Pbe(7)]-alpha-MSpI (NDP-alpha-MSH) agoniat molecules for MC4R
binding.
MC4R is a receptor for alplia-MSH. a.lpha-MSH bincling to recombinantly expressed MC4R in HEK293 cell membranes (Perkin Elmer Life and Analytical Sciences, poston, MA) was examined by competive binding sasays in which increasing amounts of unlabeled MC4R agonists (positive controls) and the p'c domain of a human ankibody (negative control) were added to assay cocktails containing ['2'I] NDP-alpba-MSH. The unlabeled MC4R agonists were melanotan II(MTII; an alpha MSH analog), alpha-MSH, and NDP-alpha-MSH. Alpha-MSIH mimetibody binding to MC4R was st.abie after two weeks of storage at 4 C, -20 C, and -80 C in PBS (phosphate buffered saline) as assessed by competive binding assays.

[0068] Competivive binding assays were performed using Scintiltation Proximity Assays(D (Amershani Biasciencea Corp, Piscataway, NJ) as directed by the assay manufacturer. Assay cocktails contained ["'1]-NDP-alpha-MSII at F:C80, i.e., -0.5 nM, 0.1 pg of MC4R membranes, 1 mM MgSO4, 1.5 mM CaClZ, 25 in11d Hepes, 0.2% BSA, mM 1,10-phenth:roline, an assay manufacturer reeommanded quantity of protease inhibitor cocktail (R.oche Diagnostics Corp., Indianapolis, IN) and SPA beads. Light emission from Scintillation Proximity AssayS beads was measured with a Packard Top Count NX'C
Instrument (Perkin Elmer IJife and Analytical Sciences, Boston, MA) for 5 minutes.

Other on'u a om 1o s and Fusions [0069] Other exemplary rpelanocyte-stimulating honnone peptides include, but are not limited to, 3-MSH and (-MSH, fragments of such peptides, homologs of such peptides, mimetics (peptide or non-peptide) of such peptides, fusion proteins comprising such pepti des, conjugates of such peptides, and any pharmaceutical salts of such peptides.
Systems and Methods [0070] In one embodiment, the method of the present invention is particularly useful for treating obesity. The methods and compositio.ns of the invention can be used to reduce, ameliorate, or prevent obesity in a subject that suffers from obesity, or is at risk of developing obesity.

[0071] According to one aspect of the invention, implantable drug infusion pumps are used to provide site specif.~c and/or sustained delivery of the appetite suppressing agents to a tocalized region of the subject. The pumps can be used for the continuous or periodic delivery over relatively long periods of time. Tlie implantable pumps ensures that the appetite suppressing agent is delivered to the target site in the central nervous system (e.g., the brain), at a concentration and for a duration effective to cause a therapeutic effect.

[0072] A variety of known implantable drug infusion pumps can be usedto doliver the appetite suppressing agent according to the present invention. Suitable infusion pumps must be capable of delivering a dnig to a site within the central nervous system, such as the brain or the intrathecal space, over an extend period of time. Suitable pumps include those that continuously deliver the drug at a selected flow rate, those that deliver the drug at a flow rate according to a programmed or programmable protocol, those that deliver the drug based on sensed physiological parameters ancl those that deliver the drug at an adjustable flow rate. Exemplary infusion pumps include those having osmotic pumps, pressure driven pumps, motorized pumps, and others known to those skilled in the art.
[0073] An impklntable delivery pump according to the present invention may include, for example, an implantable osmotic delivery pump as desen'bed in U.S. Pat. Nos.
5,728,396, 5,985,305, 6,113,938, 6,132,420, 6,156,331, 6,375,978, 6,395,292, the conten'ts of each of which are incorporated herein in their entirety by reference. An implantable pump according to the present invention may also include a regulator-type imp]antable pump that provides constant flow, adjustable flow, or programmable flow of appetite suppressing agent formulations. Examples of non-osmotic implantable pumps that may be included in an implantable pump of the present invention include those pumps described in U.S. Pat. Nos. 5,713,847, 5,368,588, 6,436,091, 6,447,522, and 6,248,112, the contents of each of which arc incorporated herein in their entirety by reference. Other implantable pumps are disclosed in U.S. Pat. Nos. 5,034,229, 5,057,318, and 5,110,596, tlle contents of which are incorporated herein by reference. Ftutber examples of implantable pumps are described in U.S. Pat. Nos. 6,283,949, 5,976,109, 5,836,935, 5,511,355, which arc incorporated herein by reference.

17ormularions [0074] The appetite suppressing agent can be formulated as a stable suspension suitable for delivery by an implantable infu,gion pump to a site within the central nervous system.
In particular, the appetite suppressing agent can be formulated such that it is stable at ambient and physiological temperatures. In one embodiment, the appetite suppressing agent is a melanocortin protein or peptide fragmont thereof. Peptides and proteins are naturally active in aqueous environments, however, peptide and protein stability is often a problem in aqueous formulations used for long durations of time at auibient or physiological temperatures. Peptides and proteins are unstable ancl tend to degrade via a number of inechanisms, including deamidation, oxidation, hydrolysis, disulfide interchange, and racemization.

(0075) One method for providing peptide and protein formulations that are stable over time at ambient or physiological temperatures, conjugating the peptide or protein to an antibody a:r an antibody fragment, to provide a stable antibody-appetite suppressing peptide conjugate, as described herein and in more detail in U.S. Application No.
60/637,818, incorporated herein by reference. This appetite suppressing antibody-peptide conjugate (rnimetibody) can then be formulated into a stable suspension for delivery by an implantable pump.

[0076] Suitable stablo suspensions of the appetite suppressing peptide or mimetobody can be fonnulated in an excipient such vvater, saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, polyethylene glycol-containin$
physiologically balanced salt solutions, and other aqueous, physiologically balanced, salt solutions. Nonaqueous vehicles, such as fixad oils, sesame oil, ethyl oleate, o:r triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
Preferably MSH mimetibodies would ba in formulations of Phosphate buffer solution or saline. Concentrations of up to 30 mg/ml have been observed in both solutions without any observable ebange in integrity and activity of the protein.

[0077] One skilled in the art will recognize that excipients can also contain minor amounts of additives, such as substances that enliarlce isotonicity and chemical stability, or buffers. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, fonnalin and benzyl alcohol. Stand2ird, fonnulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can co:m.prise clextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration [0078] Other techniques for producing a stable formulation for delivery via an implantable infusion pump, according to the invention, include suspending the appetite suppressing agent in a vehicle such as a non-aqueous vehicle, an anhydrous vehicle, an aprotic vehicle, a hydrophobic vehicle, a non-polar vehicle, a tion-aqueous vehicle, a protic vehicle, an anhydrous psuedoplastic and thixotropic oleaginous vehicle, a liposomal vehicle, and a cationic lipid vehicle.

[0079] Maintaining a substantially uniform dispersion of appetite suppressing agent over time facilitates controlled delivery of the appetite suppressing agent from an implanted pump. It is important that the appetite suppressing agent remain uniformly dispersed within a suspension that is loaded into an implantable infusion pump. A lack of uniform dispersion may result in a non-uniform amount of appetite suppressing agent being delivered to the target site, which may cause the amount of appetite suppressing agent delivered from the implanted pump to exceed recommended dosing regimons or, alternatively, aauae the amount of appetite suppressing agent delivered to fall below therapeutic levels.

[0080] In one embodiment, a substantially uniform dispersion of appetite suppressing agent is maintained by incorporating high viscosity material within the suspension.
Exemplary viscosity enhancing materials include polymers, such as olyvinylpyrrolidone, may be used to provide suspension vehicles that not only allow the fornzulation of the appetite suppressing agent suspensions that are stable over time, bu.t also offer the viscosity required to maintain a substantially uniform dispersion of agent. To achieve high viscosity vehicles using polymer materials, the polymer may be dissolved in a non-aqueous solvent to create single phase, viscous solution.

[0081] The formulations may comprise the appetite suppressing agent with about 0.1 /õ to 90% by weight of the agent, about 0.1 % to about 50%, about 0.1 % to about 25%, about 0.1 % to about 10%, and about 0.1% to 1.0% by weight of the agent.

[0082] In accordance with the present invention, a suitable or effective single dose size is a dose that is capable of causing a measurable change in the body weight (e.g., a decrease in body weight) of a subject when administered one or more times over a suitable time -26-=

period. Doses can vary depending upon the condition of the subject being treated, including the apparent cause of the body weight problem and/or any other related or non-related hea]th factors experienced by a particular subject.

[0083] In one embodiment, the invention comprises delivering an appetite suppressing agent, e.g., a melanocortin at a dose, concentration, and for a time su ..ff'ic:ient to cause a measurable change in the body weight or mass of the subject. The dose of the melanocortin can be between about 0.1 g and about 100 mg per kilogram bod.y weight of the subject; between about 0.1 g and about 10 mg per kilogram body weight of the subject; between about 0.1,ug and about I pg per kilogram body weight of the subject; and between about l g and about 10 mg per kilogram body weight of, the subject. A
typical daily dose for an adult hnman (i.e., a 75 kg human) is from about 1 milligram to about 100 milligrams. In practicing this method, the appetite suppreasing agent or tlierapeutic formulation containing the appetito suppressing agent can be administered in a single daily dose or in multiple doses per day. This treatment method may require administration over extended periods of timo. Tho amount per administered dose or the total amount adniinistered, will be determined by the physician and will depend on such factors as the mass of tho subject, the age and general health of the subject and the tolerance of the subject to the compound.

[00$4] In one embodiment, the appetite suppressing agent can be delivered alone or in combination with another agent, such as another appatite suppressing agent, e.g., leptin and/or neuropeptin Y. In another embodiment, the appetite suppressing agent can be delivered in combination with another therapeutic agent, such as a pain controlling agent.
[0085] One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are oxpressly incorporated herein by reference in thair entirety.

[0086] The prefer sites in the brain would be uypothalamus, specifically paraventricular nucleus and ventro-medical nucleus of hypothalamus. In addition, nucleus of solitary tract would be also of benefit. Above listed niaclei are the known centers for regulation of energy balance that have high level of expression of MC4 and MC3 receptors.

Claims (38)

1. A method of suppressing appetite comprising-implanting a drug infusion pump into a site in a subject; and delivering a stable suspension of an effective dosage of an appetite suppressing agent to a site in a central nervous system of the subject, wherein the appetite suppressing agent binds to a target receptor on a neural cell in the central nervous system and modifies the receptor function to suppress appetite.

2. The method of claim 1, wherein the appetite suppressing agent is a melanocyte-stimulating hormone (MSH).

3. The method of claim 2, wherein the melanocyte-stimulating hormone is alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide.

4. The method of claim 3, wherein the alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide is conjugated to an antibody or antibody fragment.

5. The method of claim 1, wherein the target receptor is a melanocortin receptor selected from the group consisting of melanocortin-1 receptor (MC1R), melanocortin-2 receptor (MC2R), melanocortin-3 receptor (MC3R), melanocortin-4, receptor (MC4R), and melanocortin-5 receptor (MC5R).

6. The method of claim 1, wherein the target receptor is melanocortin-4 receptor (MC4R).

7. The method of claim 1, wherein the target is melanocortin-3 receptor (MC3R).

8. The method of claim 1, wherein the site in the central nervous system is the brain.

9. The method of claim 1, further comprising delivering a second appetite suppressing agent selected from the group consisting of leptin, NPY peptide, antagonist, PYY 3-36 peptide agonist or its analog, GLP-1 peptide or its analog and MCH receptor antagonist.

10. A method for ameliorating obesity in a subject, comprising:
implanting a drug infusion pump into a site in a subject; and delivering a stable suspension of a melanocortin to a region in a central nervous system of the subject, wherein the melanocortin binds to a melanocortin receptor on a neural cell in the central nervous system and modifies the melanocortin receptor function to suppress appetite, thereby ameliorating obesity.

11. The method of claim 10, wherein the melanocyte-stimulating hormone is alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide.

12. The method of claim 11, wherein the alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide is conjugated to an antibody or antibody fragment.

13. The method of claim 12, wherein the target receptor is a melanocortin receptor selected from the group consisting of melanocortin-1 receptor (MC1R), melanocortin-2 receptor (MC2R), melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and melanocortin-5 receptor (MC5R).

14. The method of claim 10, wherein the target receptor is melanocortin-4 receptor (MC4R).

15. The method of claim 10, wherein the target receptor is melanocortin-3 receptor (MC3R).

16. The method of claim 10, wherein the region in the central nervous system is the brain.

17. The method of claim 10, further comprising delivering a second appetite suppressing agent selected from the group consisting of leptin, NY peptide antagonist, PYY 3-36 peptide agonist or its analog, GLP-1 peptide or its analog, and MCH receptor antagonist.

18. A system for suppressing appetite comprising:
an implantable drug infusion pump; and a stable suspension of an effective dosage of an appetite suppressing agent wherein the appetite suppressing agent can bind to a target receptor on a neural cell in the central nervous system and upon binding modifies the receptor function to suppress appetite.

19. The system of claim 18, wherein the appetite suppressing agent is a melanocyte-stimulating hormone (MSH).

20. The system of claim 19, wherein the melanocyte-stimulating hormone is alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide.

21. The system of claim 18, wherein the alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide is conjugated to an antibody or antibody fragment.

22. The use of a drug infusion pump implantable into a site in a subject for suppressing appetite, wherein the drug infusion pump is adapted to deliver a stable suspension of an effective dosage of an appetite suppressing agent to a site in a central nervous system of the subject, wherein the appetite suppressing agent binds to a target receptor on a neural cell in the central nervous system and modifies the receptor function to suppress appetite.

23. The use of claim 22, wherein the appetite suppressing agent is a melanocyte-stimulating hormone (MSH).

24. The use of claim 23, wherein the melanocyte-stimulating hormone is alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide.

25. The use of claim 24, wherein the alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide is conjugated to an antibody or antibody fragment.

26. The use of claim 22, wherein the target receptor is a melanocortin receptor selected from the group consisting of melanocortin-1 receptor (MC1R), melanocortin-2 receptor (MC2R), melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and melanocortin-5 receptor (MC5R).

27. The use of claim 22, wherein the target receptor is melanocortin-4 receptor (MC4R).

28. The use of claim 22, wherein the target is melanocortin-3 receptor (MC3R).

29. The use of claim 22, wherein the site in the central nervous system is the brain.

30. The use of claim 22, wherein a second appetite suppressing agent selected from the group consisting of leptin, NPY peptide, antagonist, PYY 3-36 peptide agonist or its analog, GLP-1 peptide or its analog and MCH receptor antagonist is deliverable.

31. The use of a drug infusion pump implantable into a site in a subject for ameliorating obesity in the subject, wherein said drug infusion pump is adapted to deliver a stable suspension of a melanocortin to a region in a central nervous system of the subject, wherein the melanocortin binds to a melanocortin receptor on a neural cell in the central nervous system and modifies the melanocortin receptor function to suppress appetite, thereby ameliorating obesity.

32. The use of claim 31, wherein the melanocyte-stimulating hormone is alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide.

33. The use of claim 32, wherein the alpha melanocyte-stimulating hormone (.alpha.-MSH) peptide is conjugated to an antibody or antibody fragment.

34. The use of claim 33, wherein the target receptor is a melanocortin receptor selected from the group consisting of melanocortin-1 receptor (MC1R), melanocortin-2 receptor (MC2R), melanocortin,3 receptor (MC3R), melanocortin-4 receptor (MC4R), and melanocortin-5 receptor (MC5R).

35. The use of claim 31, wherein the target receptor is melanocortin-4 receptor (MC4R).

36. The use of claim 31, wherein the target receptor is melanocortin-3 receptor (MC3R).

37. The use of claim 31, wherein the region in the central nervous system is the brain.

38. The use of claim 31, wherein a second appetite suppressing agent selected from the group consisting of leptin, NY peptide antagonist, PYY 3-36 peptide agonist or its analog, GLP-1 peptide or its analog, and MCH receptor antagonist is deliverable.