WO2014024189A1

WO2014024189A1 - Pegylated high affinity leptins with increased biological activity

Info

Publication number: WO2014024189A1
Application number: PCT/IL2013/050668
Authority: WO
Inventors: Arieh Gertler; Michal Ayalon-Soffer
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd; Biolinerx Ltd
Priority date: 2012-08-06
Filing date: 2013-08-06
Publication date: 2014-02-13

Abstract

A pegylated leptin agonist comprising a PEG moiety linked to a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild- type human leptin (D23) is substituted with a different amino acid residue that is not negatively charged or the threonine at the position corresponding to position 12 of the wild-type human leptin (T12) is substituted with a different amino acid residue that is hydrophobic or a fragment of said modified mammalian leptin polypeptide is provided.

Description

PEGYLATED HIGH AFFINITY LEPTINS WITH INCREASED BIOLOGICAL ACTIVITY

FIELD OF THE INVENTION

[0001] The present invention relates to improved mutated leptin agonists and to pharmaceutical compositions comprising them.

BACKGROUND OF THE INVENTION

[0002] An analog of human leptin, metreleptin (recombinant methionyl human leptin) is under investigation for the treatment of diabetes and/or hypertriglyceridemia, in patients with rare forms of lipodystrophy, syndromes characterized by abnormalities in adipose tissue distribution, and severe metabolic abnormalities (see for example US 8,394,765).

[0003] In order to improve the action of leptin, leptin agonists with improved affinity for the leptin receptor were developed; however, these high affinity leptin agonists had the same biological activity as unmodified wild type (WT) leptin (WO 2011/132189), and thus conferred no advantage.

[0004] There is therefore a wide felt need for improved leptin agonists for the treatment of diseases associated with aberrant leptin signaling and/or eventual use of leptin as anti-obesity agent along with other proteins like amylin or similar.

SUMMARY OF INVENTION

[0005] In some aspects, the present invention provides a pegylated leptin agonist comprising a PEG moiety linked to: (i) a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with a different amino acid residue that is not negatively charged or the threonine at the position corresponding to position 12 of the wild-type human leptin (T12) is substituted with a different amino acid residue that is hydrophobic; (ii) a fragment of said modified mammalian leptin polypeptide of (i), comprising a continuous stretch of amino acid residues corresponding to positions 12 to 42 of the wild-type human leptin, said continuous stretch comprising said different amino acid residue that is not negatively charged at the position corresponding to position 23 of the wild-type human leptin or said different amino acid residue that is hydrophobic at the position corresponding to position fragment is itself a leptin agonist; or (iii) a pharmaceutically acceptable salt of (i) or (ii).

[0006] In other aspects, the present invention provides pharmaceutical compositions comprising the above defined pegylated leptin agonist, and a pharmaceutically acceptable carrier.

[0007] In still other aspects, the present invention provides the above defined pegylated leptin agonist, or pharmaceutical compositions comprising such pegylated leptin agonists, for use in treatment of a disease, disorder or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia-related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis; or promotion of angiogenesis.

[0008] In yet other aspects, the present invention relates to methods for treatment of a disease or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia-related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis, or for promotion of angiogenesis, comprising administering to a patient in need an effective amount of the pegylated leptin agonist as defined above, or an effective amount of a pharmaceutical composition comprising such pegylated leptin agonists.

BRIEF DESCRIPTION OF DRAWINGS

[0009] Fig. 1 shows a comparison of binding of the non-pegylated D23L mutant human leptin (hL-D23L) with wild type (WT) human leptin (hL). OD, optical density; on the X-axis, log concentration (M).

[0010] Fig. 2 shows effect of 14 days repeated IP administration of pegylated human leptin (PhL) and pegylated D23L mutant human leptin (PhL-D23L) on blood glucose levels over time compared to vehicle (water for injection; WFI) administration in male ob/ob mice.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention is based on the finding that while the biological activity of a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with leucine (herein after referred to as leptin D23L (L-D23L) having an amino acid sequence as set forth in SEQ ID NO: 1) and that is pegylated, is about five-fold more effective in in vitro cell bioassays than non-mutated (wild type) pegylated leptin, while non-pegylated L-D23L has substantially the same biological activity as non-pegylated wild-type leptin (see Example 1). This was surprising, since there was no reason to expect that pegylation would affect the biological activity, and there is presently no explanation for the fact that only the activity of the mutated leptin agonist was increased, while that of the wild type leptin was unaffected.

[0012] The location of a certain amino acid residue in the proteins or fragments thereof disclosed herein is according to the numbering of the wild type human leptin as depicted in SEQ ID NO: 2 and is designated by referring to the one-letter code of the amino acid residue and its position in the wild type human leptin. Thus, for example, the aspartic acid at the position corresponding to position 23 of the wild-type human leptin, also referred to herein as D23, would be referred to as D23 also in a leptin fragment or in a homologous mammalian leptin of a different size according to alignment algorithms well known in the art of protein chemistry (e.g. SEQ ID NO: 1 has an extra alanine residue at the N-terminus; therefore D23 is found at position 24 in this sequence). A substitution of an amino acid residue at a certain position with another amino acid residue is designated by referring to the one-letter code of the amino acid residue, its position as defined above and the one- letter code of the amino acid residue replacing the original amino acid residue. Thus, for example, a substitution of D23 with leucine would be designated D23L.

[0013] WO 2011/132189, incorporated by reference as if fully disclosed herein, teaches that replacement of D23 by any amino acid not carrying a negative charge was sufficient to increase the affinity toward human Leptin Binding Domain (a soluble leptin receptor) and subsequent biological activity. The highest effect was observed with the D23L mutant in binding and cell assays. WO 2011/132189 further teaches that substitution of T12 with a different amino acid residue that is hydrophobic increases the leptin's affinity towards its receptor. Other substitutions introduced into the leptin polypeptide, optionally in addition to the substitutions of D23 and/or T12, such as L68M, S97F, S132Y, G112S, T37A and G44D, and any combination of two or more of these substitutions, also increase the affinity of leptin.

[0014] Thus, in one aspect, the present invention provides a pegylated leptin agonist comprising a PEG moiety linked to: (i) a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with a different amino acid residue that is not negatively charged or the threonine at the position corresponding to position 12 of the wild-type human leptin (T12) is substituted with a different amino acid residue that is hydrophobic; (ii) a fragment of said modified mammalian leptin polypeptide of (i), comprising a continuous stretch of amino acid residues corresponding to positions 12 to 42 of the wild-type human leptin, said continuous stretch comprising said different amino acid residue that is not negatively charged at the position corresponding to position 23 of the wild-type human leptin or said different amino acid residue that is hydrophobic at the position corresponding to position 12 of the wild-type human leptin and the amino acid sequence LDFI, wherein said fragment is itself a leptin agonist; or (iii) a pharmaceutically acceptable salt of (i) or (ii).

[0015] In certain embodiments, D23 is substituted with a hydrophobic or positively charged amino acid residue. For example, the hydrophobic amino acid residue may be selected from leucine, glycine, alanine, tryptophane, histidine or phenylalanine; and the positively charged amino acid residue may be selected from arginine or lysine. In particular, D23 is substituted with leucine.

[0016] In certain embodiments, T12 is substituted with isoleucine.

[0017] In certain embodiments, further amino acid residues of the pegylated leptin agonist are substituted as follows: (i) the leucine at the position corresponding to position 68 of the wild-type human leptin (L68) is substituted with methionine, the serine at the position corresponding to position 97 of the wild-type human leptin (S97) is substituted with phenylalanine and the serine at the position corresponding to position 132 of the wild- type human leptin (SI 32) is substituted with tyrosine; (ii) the glycine at the position corresponding to position 112 of the wild-type human leptin (G112) is substituted with serine; or (iii) the threonine at the position corresponding to position 37 of the wild-type human leptin (T37) is substituted with alanine and the glycine at the position corresponding to position 44 of the wild-type human leptin (G44) is substituted with aspartic acid.

[0018] As used herein, the term "mammal" or "mammalian" includes human mammal as well as non-human mammals. Thus, according to the present invention, the native leptin may be human leptin or a non-human mammal leptin such as, but not limited to, ovine, rat, mouse, horse and pig leptin, and the LDFI sequences represent the 39-42 LDFI sequence of human leptin or of a non-human mammal leptin. In certain embodiments, the leptin is human, mouse or ovine leptin.

[0019] The term "about" preceding numbers expressing quantities, percentages or proportions, and other numerical values recited herein, refers to a range of 10% below or above said numbers, or in the case of days, 1, 2 or 3 days before or after the recited day.

[0020] Pegylation was not expected to affect the activity of the leptin molecules because the modification was done in order to increase the circulation half-life and solubility. The reason PEG is used for these purposes is that PEG, when connected to a protein, prevents proteolytic enzymes from accessing the protein, the associated water molecules on PEG increase the solubility, and the increased molecular weight of the PEG- protein complex reduces the speed of kidney clearance.

[0021] The leptin proteins of the present invention may be pegylated by any method known in the art or, for example, but not limited to, methods disclosed in Roberts et al, 2002, and Broyer et al., 2011, both publications herein incorporated by reference as if fully disclosed herein.

[0022] The PEG moiety may be linked to the protein via a stable linkage, which is beneficial in the sense that it is appropriate for long-term storage, easy purification and availability of prefilled syringes. Another possible solution is to link the PEG moiety to the protein via a degradable linkage. The linker may be severed for example by low pH, enzymatic degradation, hydrolytic cleavage or reduction.

[0023] The linkage of the PEG molecule(s)/moietie(s) to the leptin agonist may be direct or via a spacer to decrease crowding effect, to increase reactivity, and reduce steric hindrance. The application of a spacer arm can enhance receptor binding.

[0024] Amino acid spacers such as alanine, glycine, and small peptides may be used due to their chemical versatility for covalent conjugation and biodegradability. Heterobifunctional coupling agents containing succinimidyl are also a good alternative.

[0025] The degradable linkage may be comprised within the spacer; e.g. it may comprise recognition sites for specific proteases or sites that undergo hydrolysis at a certain pH, in order to release the active leptin agonist from the PEG motility.

[0026] In certain embodiments, the PEG moiety is linked to a reactive group on the protein such as an amino, carboxyl, hydroxyl or thiol group of the protein. For example, the PEG moiety may be linked to such a reactive group in one or more lysine, arginine, aspartic acid, glutamic acid, free cysteine, serine, threonine, histidine or tyrosine residue(s) of the leptin protein, as well as the free terminal amino group or the free terminal carboxyl group, as set forth in SEQ ID NO: 1 or 2.

[0027] In certain embodiments, the PEG-moiety is linked via a lysine residue or the N- terminus. In certain embodiments, the PEG molecule comprises a functional group selected from activated esters, thioimido esters, isocyanates, chlorotriazines, aldehydes or ketones.

[0028] In certain embodiments the functional group of the PEG is an aldehyde, such as propionylaldehyde .

[0029] In certain embodiments, the synthetic leptin agonist has a variable number of polyethylene glycol (PEG) molecules attached thereto, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 PEG molecules/moieties per protein. In particular, one or two PEG moieties are attached to each protein molecule.

[0030] In certain embodiments, the leptin agonist is pegylated at its N-terminal amino group. In certain embodiments, at least 50, 60, 70, 80, 85, 90, 95, 99 or 100% of the leptin agonists in a solution, or a certain batch of preparation, are pegylated at the N-terminal amino group, and in particular 85% of the leptin agonists. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20% of the leptin agonists in a solution, or a certain batch of preparation, are pegylated at two locations. In certain embodiments, less than 1% of the leptin agonists in a solution, or a certain batch of preparation, is non-pegylated.

[0031] The PEG molecular weight has a direct impact on the activity; in general, higher molecular weight PEG conjugates tend to have lower in vitro activity but have higher in vivo activity due to the improved pharmacokinetic profile.

[0032] Many of the important benefits of PEGylation can be controlled by proper conjugation of various molecular weight PEGs to the protein at specific locations on the protein's surface.

[0033] PEG of a molecular weight of about 5 kDa to 40 kDa is suitable for this purpose, in particular lOkDa to 30 kDa or 15 kDa to 25 kDa. More specifically, PEG molecules/moieties of 5, 6, 7, 8, 9, 10 , 12, 14, 16, 18 or 20 kDa may provide the desired effect. The PEG moiety may be linear, star- shaped or branched. In certain embodiments, the PEG moitety is a linear 20 kDa polymer.

[0034] In certain embodiments, the pegylated leptin agonist according to the present invention comprises a linear PEG moiety linked to the N-terminal amino group of a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with leucine.

[0035] Also included in the scope of the invention are salts of the pegylated mammalian leptin polypeptides of the invention. As used herein, the term "salts" refers to both acid addition salts of amino groups and to salts of carboxyl groups of the peptide molecule. Examples of acid addition salts are, without being limited to, the mesylate salt, the maleate salt, the fumarate salt, the tartrate salt, the hydrochloride salt, the hydrobromide salt, the esylate salt; the p-toluenesulfonate salt, the benzoate salt, the acetate salt, the phosphate salt, the sulfate salt, the citrate salt, the carbonate salt, and the succinate salt. Additional pharmaceutically acceptable salts include salts of ammonium (NH4⁺) or an organic cation derived from an amine of the formula R4N⁺, wherein each one of the Rs independently is selected from H, C1-C22, preferably C1-C6 alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec -butyl, isobutyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl, n-hexyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, lithium, potassium, ammonium, ferric or zinc salts, and the like. Such salts are preferably used to modify the pharmaceutical properties of the polypeptide insofar as stability, solubility, etc., are concerned.

[0036] It has further been found in accordance with the present invention that daily intraperitoneal (IP) administration of the mutated pegylated leptin as defined herein above (herein after referred to as PL-D23L or PhL-D23L for the human leptin mutant) for 14 consecutive days in ob/ob mice (mice having a nonsense mutation in the leptin gene leading to a null allele; a mutant mouse that eats excessively and becomes profoundly obese) significantly decreased body weight and food consumption as compared to vehicle- treated group. It was further found that repeated administration of PhL-D23L significantly decreased blood glucose levels both prior to glucose challenge and following Oral Glucose Tolerance Test (OGTT; see Example 2).

[0037] Thus, in some aspects, the present invention provides a pegylated leptin agonists as defined herein above, or pharmaceutical compositions comprising such pegylated leptin agonists, for the preparation of a medicament for treatment of a disease, disorder or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia-related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis, or for promotion of angiogenesis (it has been shown that leptin has a role in angiogenesis: For example, Park et al. (2001) showed that leptin-receptor expression is increased in the intima of neorevascularized regions and using the rat corneal angiogenesis assay, leptin elicited a comparable sensitivity of angiogenic activity to those of vascular endothelial growth factor) .

[0038] In other aspects, the present invention provides a pegylated leptin agonists as defined herein above, or pharmaceutical compositions comprising such pegylated leptin agonists for use in treatment of a disease, disorder or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia-related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis, or in promotion of angiogenesis.

[0039] In certain embodiments, the disorder is obesity.

[0040] The phrase " disease, disorder or condition in which aberrant leptin signaling is implicated" is used herein interchangeably with the phrase " disease, disorder or condition associated with aberrant leptin signaling", and should be understood as a disease, disorder or condition caused by aberrant production or homeostasis of leptin, aberrant receptor expression or function or downstream signaling; or a disease, disorder or condition which symptoms may be improved by supplement of leptin activity.

[0041] Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0042] The following exemplification of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results.

[0043] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

[0044] Methods of administration of the pharmaceutical compositions of the invention include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local.

[0045] The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.

[0046] So far leptin therapy was used in limited cases in which genetic deficiency of leptin was identified (Bluher et al. 2009) or on an experimental basis in human lipostrophy (Chong et al. 2010). However a continuous effort to utilize leptin as an anti-obesity drug continues and despite the failures a recent report describing successful combination of leptin and amylin therapy was published (Turek et al. 2010). Another report showed that pretreatment of mice with a chemical chaperone such as buphenyl (4-PBA) or tauroursodeoxycholic acid (TUDCA) increased leptin sensitivity (Ozcan et al., 2009).

[0047] Also, in light of a recent report showing that in mice with type 1 diabetes treated with leptin in combination with insulin blood sugar fluctuated less, cholesterol levels were lower and there was less body fat deposition than in mice with type 1 diabetes treated with insulin alone (Wang et al., 2010), the leptin agonists of the present invention may be used in conjunction with insulin or other agents mediating glucose homeostasis in the treatment of type 1 diabetes.

[0048] Thus, the pharmaceutical composition may comprise one or more further active agents. For example, for the treatment of diabetes type I, the pharmaceutical composition may comprise insulin in addition to leptin agonist and for the treatment of obesity the pharmaceutical composition may comprise an amylin agonist. The human form of amylin has the amino acid sequence as set forth in SEQ ID NO: 3, with a disulfide bridge between cysteine residues 2 and 7. Both an amidated C-terminus and the disulfide bridge are necessary for the full biological activity of amylin, and therefore any amylin analog must comprise at least these two features. Furthermore, the analog should be devoid of amyloidogenic activity, such as rat amylin. In particular, the amylin analog may be pramlintide acetate (developed by Amylin Pharmaceuticals).

[0049] For the treatment of obesity the pharmaceutical composition may comprise, in addition to leptin agonist, a chemical chaperone such as buphenyl (4-PBA) or tauroursodeoxycholic acid (TUDCA) in addition to the leptin agonist.

[0050] Thus, in certain embodiments, the treatment of obesity may be combined with the further administration to the patient of an amylin analog such as SYMLIN^® (pramlintide acetate) or a chemical chaperone such as buphenyl (4-PBA) or tauroursodeoxycholic acid (TUDCA).

[0051] In certain embodiments, the method for treatment of type 1 diabetes comprising administering to a patient in need an effective amount of the pegylated leptin agonist as defined above, is combined with the further administration to the patient of insulin.

[0052] The amylin analog, chemical chaperone or insulin may be provided concomitantly with the pegylated mutated leptin analog or sequentially.

[0053] The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Example 1: Comparison of Non-Pegylated and Pegylated Wild Type Human Leptin and its D23L Mutant.

[0054] The recombinant mutated leptins were expressed with an extra Met- Ala (methionine is cleaved by the bacteria) at the N-terminus.

[0055] The D23L leptin mutant (L-D23L) and its pegylated form were prepared as described for similar leptin antagonists in WO 2011/132189. In short, mPEG-propionyl- ALD 20 kDa (Jenkem Technology USA Inc., Allen, TX) was used for pegylation under conditions in which the N-terminal amino group is preferentially pegylated (ALD is short for aldehyde). 150 mg of human leptin (hL) was dissolved in 111 ml of 0.1 M Na- Acetate buffer (pH 5) and centrifuged at 12000 rpm 10 min to remove the insoluble material. Then 0.2 M of NaBH3CN (2.7 ml) was added and the dissolved protein was conjugated with 1.5 g mPEG-propionyl-ALD 20 kDa that was dissolved in 15 ml of 1 mM HC1. After 20 hours of stirring at 4°C, 160 μΐ of acetic acid (17 M) was added. The solution was stirred for a few seconds, diluted with 1 L double distilled (dd)H₂0 and applied at maximal flow rate (400-500 ml/h) onto a SP-Sepharose column (20-ml bead volume), pre-equilibrated with 10 mM Na- Acetate, pH 4. The column was then washed with 400 ml of 10 mM Na- Acetate, pH 4 and the pegylated protein was eluted in 10 mM Na- Acetate, pH 5, containing 50 and 75 mM NaCl. Fractions containing the pegylated protein as determined by gel filtration on analytical Superdex 200 column (10/30 cm) were pooled, dialyzed against NaHC0₃ to ensure a 2: 1 protein-to-salt ratio and lyophilized. Protein concentrations were determined by absorbance at 280 nm using an extinction coefficient (for 0.1% solution of pegylated protein) of 0.200 mg/ml for human leptin. Those values apply to the protein part of the pegylated product.

[0056] In the case of the leptin antagonists of WO 2011/132189, the final preparation of PEG-antagonists was pure by SDS-polyacrylamide gel electrophoresis (PAGE) criteria and contained ~ 9 % of double pegylated antagonists, 85 % of monopegylated antagonists and less than 1% of non-pegylated antagonists.

[0057] Binding assay - Biotinylated mouse leptin served as the ligand in all competitive experiments and the respective mouse leptin or mouse or human leptin antagonist muteins as competitors. Soluble human leptin receptor (hLBD) was used as the receptor source. Polystyrene 96-well microtiter plates were coated overnight at 4°C with 100 μΐ of 40 pM hLBD in phosphate buffered saline (PBS) pH 7.4. Wells were then washed one time with PBST (PBS containing 0.05 % Tween 20) and blocked with PBS containing 3% skim milk for two hours in room temperature. All further incubations were carried out at room temperature. Wells were washed one time with PBST and incubated with different concentrations of un-labeled pegylated or non-pegylated leptins (50 μΐ/well) for 30 min and then 50 μΐ of 62.5 pM of biotinylated mouse leptin were added to each well for another two hours. Then the wells were washed three times with PBST and incubated with 1:30,000 streptavidin-horse-radish-peroxidase (HRP) in PBS containing 1 % Tween 20 for one hour. Subsequently the wells were washed three times with PBST and the reaction was quantified in 450 nm by microplate reader ELISA Plate Reader ELx808 - Table 1. Leptin activity (EC₅₀ in pM)

¹ increase in activity of hL-D23L compared to WT human leptin (hL)

increase in activity of PhL-D23L compared to pegylated WT human leptin (PhL)

Bio-Tek Instrument Inc. (Winooski, VT, USA) using 3,3',5,5'-Tetramethylbenzidine (TMB) according to manufacturer's instructions.

[0058] BAF/3 proliferation assays - The proliferation rate of leptin- sensitive BAF/3 cells stably transfected with the long form of human leptin receptor was used to estimate agonistic activity of pegylated or non-pegylated leptins and leptin muteins as described previously (Niv-Spector L, Gonen-Berger D, Gourdou I, Biener E, Gussakovsky EE, Benomar Y, Ramanujan KV, Taouis M, Herman B, Callebaut I, Djiane J, Gertler A (2005) Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists. Biochem J 391:221-30; Salomon G, Niv-Spector L, Gussakovsky EE, Gertler A (2006) Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists. Protein Expr Purif 47: 128- 36), incorporated by reference as if fully disclosed herein.

Results. Pegylated human D23L leptin (PhL-D23L) is about 5 fold more effective in in vitro cell Baf/3 bioassay than non-mutated human pegylated leptin (PhL), while the respective non pegylated species (hL-D23L and hL, respectively) have almost Table 2. Average fold increase in leptin activity (mean ± SEM) of seven or nine experiments with non pegylated and pegylated leptin (hL) and its D23L mutant

the same activity (Tables 1 and 2). This is startling in view of the fact that the binding of the non-pegylated D23L mutant (IC50= 3.26-10) is about 16-fold stronger compared to wild type leptin ((IC50= 4.83-9) (Fig. 1).

Example 2: Evaluation of PhL-D23L Effect on Body Weight and Glucose Levels in ob/ob Mice Following Once Daily IP Administration for 14 Days.

2.1. Test item

[0059] Species/Strain: ob/ob mice; Source: Harlan Laboratories Israel, Ltd.; Gender: Male; Group size: n=6; Number of groups: 7; Total number of animals: 42; Body Weight: about 45 gr + 15% at study initiation.

[0060] Animals Health: The health status of the animals used in this study was examined on arrival and during the entire study. Only animals in good health were placed on study.

[0061] Acclimation: At least 5 days.

[0062] Housing: During acclimation and throughout the entire study duration, animals were housed within a limited access Individually Ventilated Cage (IVC) rodent facility and kept in groups of 6 animals/cage in filtered polypropylene cages.

[0063] Food and Water: Animals were provided diet ad libitum and free access to drinking water, supplied to each cage via polyethylene bottles with stainless steel sipper tubes.

[0064] Identification: Animals were given a unique animal identification ear tag number. This number also appears on a cage card, visible on the front of each cage. The cage card also contains the study number, group number, dose level, route of administration, gender, strain and all other relevant details as to treatment group and dose level.

[0065] Randomization: During the acclimation period, animals were assigned randomly to the various test groups according to their initial body weight, when the mean body weight in each group should be similar. [0066] Termination: Animals were euthanized by C0₂ asphyxiation at scheduled termination of the study.

2.2 Study design

[0067] Test Groups 1-7 (six animals per group) are injected i.p. a volume of 5 ml/kg. The dosing regimen is 1 injection daily during 14 successive days¹. Glucose levels following OGTT 2 is measured at -15, 03 15, 30, 60 and 120 minutes.

2.3 Experimental procedures

[0068] The objective of this study was to assess the effect of PEG-Leptin D23L (PhL- D23L) on body weight and glucose levels following repeated administration by intraperitoneal (IP) injection once daily for 14 successive days in male ob/ob mice.

[0069] The test item, reference compound and vehicle were injected intraperitoneally once daily at the same time (in the morning, when the time interval between the doses is 24 hours) for 14 successive days to 7 groups of n=6 mice. One group of n=6 mice acted as the vehicle control group.

[0070] OGTT-glucose monitoring: All animals were fasted overnight before the oral administration of glucose (2gr/kg) on day 14. Oral Glucose Tolerance Test was performed 15 min following the last dosing session on day 14.

[0071] Glucose baseline levels were evaluated using a glucometer immediately before dosing (15 min prior to glucose challenge). Then, blood glucose levels were determined immediately before glucose challenge (time 0), 15, 30, 60, and 120 minutes post-challenge for all groups.

[0072] At the end of the 14-day study period, all animals were subjected to an additional 14-day recovery period.

2.4 Treatment

[0073] Route of administration: The test item, PhL-D23L, reference compound (pegylated human wild-type leptin; PhL) and vehicle control item (water for injection; WFI) were administered by intraperitoneal injections.

¹ All animals were subjected to a 14 days recovery period

² Time 0 refers to glucose challenge administered by oral gavage (2 gr/kg)

³ Glucose levels were measured immediately prior to glucose challenge • Dose level:

• Group 1 (pegylated human wild- type leptin; PhL): 1 mg/kg

• Group 2 (pegylated human wild-type leptin; PhL): 0.1 mg/kg

• Group 3 (PhL-D23L): 1 mg/kg

• Group 4 (pegylated human wild-type leptin; PhL): 0.3 mg/kg

• Group 5 (vehicle): 0 mg/kg

• Group 6 (PhL-D23L): 0.3 mg/kg

• Group 7 (PhL-D23L): 0.1 mg/kg

[0074] Volume dosage: All formulations were administered at a constant dose volume of 5 ml/kg. The volume dosage was adjusted according to determined body weights.

[0075] Frequency and duration: All treated animals were subjected to once daily IP administration for 14 consecutive days.

2.5 Observation and examination:

[0076] Clinical Signs: Throughout the entire study period, careful clinical examinations were carried out and recorded immediately following dosing up to one hour post-dosing.

[0077] Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions (e.g. diarrhea) and autonomic activity (e.g. lacrimation, salivation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of bizarre behavior, tremors, convulsions, sleep & coma were also observed and recorded.

[0078] All animals were observed for morbidity and mortality twice daily throughout the entire study period.

[0079] Body Weights: Determination of individual body weights of all animals was initially carried out at the randomization procedure, shortly before the 1st dosing session and thereafter once daily before dosing. The dosing volume was adjusted according to the latest body weight determination.

[0080] At the end of the dosing period, all animals were subjected to 14 days recovery period. Body weight was measured daily during the recovery phase at the same time during the day as in the dosing phase.

[0081] Food consumption: Measurements of estimated food consumption per cage were initially carried out during the acclimation period (prior to the 1st dosing session) and thereafter twice weekly. Food consumption was measured twice weekly during the 14 days recovery phase.

Table 3: Mortality observed in male ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin (PhL) and WFI once daily for 14 consecutive days.

(a) n=6 animals

Table 4: Clinical signs observed in male ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin (PhL) and WFI once daily for 14 consecutive days.

n=6 animals

NAD= No abnormality detected Table 5: Mean (+SD) group body weights in male ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin (PhL) and WFI once daily for 14 consecutive days.

2.6 Results:

[0082] Mortality (Table 3): No mortality was observed in any of the animals throughout the dosing period, however one animal (#13) from group # 3 (treated with PhL-D23L at dose level of 1 mg/kg) was found dead during the recovery phase (recovery day 3).

[0083] Clinical signs (Table 4): No abnormal clinical signs were observed in any of the animals throughout the study period.

[0084] Body weights (Tables 5 and 6): The mean group body weight in male ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin (PhL) and WFI once daily for 14 consecutive days is shown in Table 5 (for reference, the average weight of a normal laboratory mouse is about 20g). The weight gain of PhL-D23L-treated animals (Table 6; groups 3, 6 and 7) was significantly lower (p<0.01) than those of the vehicle control treated animals (group 5) at all doses tested from day 1 of the study until the termination of the dosing phase on Day 14. In other words, the PhL-D23L-treated mice actually lost weight, while the vehicle control treated mice gained weight. PhL-D23L- treated mice lost statistically significant more weight than PhL-treated mice.

Table 6: Individual and mean group body weights gain values in ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin (PhL) and WFI once daily for 14 consecutive days

** p<0.01 vs. group 5 (WFI)

¥ p<0.05 group 6 vs. group 4

** p<0.01 group 7 vs. group 2

& p<0.05 group 1 vs group 3

[0085] The mean group body weight gain of PhL-D23L-treated animals at 0.1 mg/kg was significantly lower (p<0.01; group 7 vs. group 2) than those of the pegylated human leptin-treated animals at the same dose level.

[0086] The mean group body weight gain of PhL-D23L-treated animals at 0.3 mg/kg was significantly lower (p<0.05; group 6 vs. group 4) than those of the pegylated human leptin-treated animals at the same dose level.

[0087] The mean group body weight gain of PhL-D23L- treated animals at 1 mg/kg was significantly lower (p<0.05; group 1 vs. group 3) than those of the pegylated human leptin-treated animals at the same dose level. Table 7: Daily food consumption per group in male ob/ob mice, following IP administration of PhL-D23L, pegylated human leptin

(PhL) and WFI once daily for 14 consecutive days

[0088] Food consumption (Table 7): The mean food consumption of PhL-D23L- treated animals at all doses tested was reduced compared to pegylated human leptin- treated animals over the course of the study. The lowest dose of PhL-D23L (0.1 mg/kg) exhibited the greatest reduction in food consumption compared to pegylated human leptin at similar dose level.

[0089] Blood glucose levels (Table 8 and Fig. 2): PhL-D23L and pegylated human leptin (PhL) were evaluated for in vivo efficacy in the regulation of glucose homeostasis using an oral glucose tolerance test on dosing day 14. Blood glucose levels of overnight- fasted ob/ob mice rose quickly after oral administration of a glucose challenge (2g/kg). However, this increase in blood glucose levels was significantly reduced in both PhL- D23L-treated and PhL-treated animals at all doses levels tested.

[0090] This decrease in glucose levels was significant (p<0.01) at all times points tested following glucose challenge (15, 30, 60, and 120 minutes post glucose challenge), as compared to vehicle-treated group.

[0091] Except of group 2 (animals dosed with PhL at 0.1 mg/kg once daily for 14 days), all PhL-D23L and PhL-treated group at all dose levels tested displayed significantly decreased blood glucose levels (p<0.01) prior to glucose challenge (at time point -15 min), meaning that repeated administration of both test item for 14 consecutive days significantly decreased high glucose levels exhibited by ob/ob mice treated with vehicle.

[0092] Furthermore, PhL-D23L-treated animals at dose level of 0.1 mg/kg (group 7) displayed significantly reduced blood glucose levels (p<0.01) 15 minutes prior to OGTT Table 8. Blood glucose levels in male ob/ob mice measured on dosing day 14, following IP administration of PhL-D23L and pegylated human leptin (PhL) and WFI once daily for 14 consecutive days

[0093] compared to PhL-treated animals at similar dose level (group 2), suggesting that repeated administration of PhL-D23L showed improved efficacy over pegylated human leptin in reducing glucose levels in ob/ob mice.

[0094] Conclusion: Once daily administration of PhL-D23L at dose levels of 0.1, 0.3 and 1 mg/kg for 14 consecutive days in ob/ob mice significantly decreased body weight and food consumption as compared to vehicle-treated group. Repeated administration of PhL-D23L significantly decreased blood glucose levels both prior to glucose challenge and following OGTT. Furthermore, PhL-D23L exhibited improved efficacy as compared to pegylated human leptin at similar dose levels.

Claims

1. A pegylated leptin agonist comprising a PEG moiety linked to:

(i) a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with a different amino acid residue that is not negatively charged or the threonine at the position corresponding to position 12 of the wild-type human leptin (T12) is substituted with a different amino acid residue that is hydrophobic;

(ii) a fragment of said modified mammalian leptin polypeptide of (i), comprising a continuous stretch of amino acid residues corresponding to positions 12 to 42 of the wild-type human leptin, said continuous stretch comprising said different amino acid residue that is not negatively charged at the position corresponding to position 23 of the wild-type human leptin or said different amino acid residue that is hydrophobic at the position corresponding to position 12 of the wild-type human leptin and the amino acid sequence LDFI, wherein said fragment is itself a leptin agonist; or

(iii) a pharmaceutically acceptable salt of (i) or (ii).

2. The synthetic leptin antagonist of claim 1, wherein D23 is substituted with a hydrophobic or positively charged amino acid residue.

3. The synthetic leptin antagonist according to claim 2, wherein the hydrophobic amino acid residue is selected from leucine, glycine, alanine, tryptophane, histidine or phenylalanine; and the positively charged amino acid residue is selected from arginine or lysine.

4. The synthetic leptin antagonist according to claim 3, wherein D23 is substituted with leucine.

5. The synthetic leptin antagonist according to claim 1, wherein T12 is substituted with isoleucine.

6. The synthetic leptin antagonist according to claim 1, wherein further amino acid residues are substituted as follows:

(i) the leucine at the position corresponding to position 68 of the wild-type human leptin (L68) is substituted with methionine, the serine at the position corresponding to position 97 of the wild-type human leptin (S97) is substituted with phenylalanine and the serine at the position corresponding to position 132 of the wild- type human leptin (SI 32) is substituted with tyrosine;

(ii) the glycine at the position corresponding to position 112 of the wild-type human leptin (G112) is substituted with serine; or

(iii) the threonine at the position corresponding to position 37 of the wild-type human leptin (T37) is substituted with alanine and the glycine at the position corresponding to position 44 of the wild-type human leptin (G44) is substituted with aspartic acid.

7. The pegylated leptin agonist according to claim 1, wherein said leptin agonist is pegylated at its N-terminal amino group.

8. The pegylated leptin agonist according to claim 1, wherein said PEG moiety has a molecular weight of about 20 kDa.

9. The pegylated leptin agonist according to claim 8, comprising a PEG moiety linked to the N-terminal amino group of a modified mammalian leptin polypeptide that is identical to wild-type mammalian leptin polypeptide, with the exception that the aspartic acid at the position corresponding to position 23 of the wild-type human leptin (D23) is substituted with leucine.

10. A pharmaceutical composition comprising a pegylated leptin agonist according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

11. The pegylated leptin agonist according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 10, for use in treatment of a disease, disorder or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia-related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis; or promotion of angiogenesis.

12. The pegylated leptin agonist or the pharmaceutical composition according to claim 11, for use in treatment of obesity.

13. The pegylated leptin agonist or the pharmaceutical composition according to claim 11, for use in combination with an amylin analog such as pramlintide acetate or a chemical chaperone such as buphenyl (4-PBA) or tauroursodeoxycholic acid (TUDCA) in treatment of obesity.

14. The pegylated leptin agonist or the pharmaceutical composition according to claim 11, for use in combination with insulin in treatment of type 1 diabetes.

15. A method for treatment of a disease, disorder or condition in which aberrant leptin signaling is implicated, selected from the group consisting of obesity, hyperphagia- related syndromes, type 1 diabetes, metabolic syndrome, hypertriglyceridemia and atherosclerosis; or for promotion of angiogenesis, comprising administering to a patient in need an effective amount of the pegylated leptin agonist according to any one of claims 1 to 9.

16. The method according to claim 15, for treatment of obesity.

17. The method according to claim 15 for treatment of obesity, further comprising administering to said patient an amylin analog such as pramlintide acetate or a chemical chaperone such as buphenyl (4-PBA) or tauroursodeoxycholic acid (TUDCA).

18. The method according to claim 15, for treatment of type 1 diabetes, further comprising administering insulin to said patient.