Method of treating obesity in a companion animal comprising administering a modified canine leptin polypeptide

Abstract

Modified canine leptin polypeptides and formulations and uses thereof, are provided including polyethylene glycol (PEG) modified canine leptin polypeptides, wherein the PEG moiety is covalently attached to a para-acetyl-phenylalanine (pAF) residue of the polypeptide, and related compositions and methods useful in treating companion animal obesity and other leptin-related disorders.

Claims

1. A method of treating obesity in a companion animal comprising administering to the companion animal in need thereof an effective amount of a compound, the effective amount is in a range of 0.3 mg/kg to 1.2 mg/kg for at least once a week for five weeks, the compound comprises a polypeptide of SEQ ID NO: 1 and a polyethylene glycol (PEG) moiety, wherein the PEG moiety is covalently attached to a para-acetyl-phenylalanine (pAF) at residue 112 of the polypeptide of SEQ ID NO: 1.

2. The method of treating obesity in a companion animal of claim 1, wherein said PEG moiety is -methyl--amimooxyethylcarbamyl, polyoxyethylene.

3. The method of treating obesity in the companion animal of claim 1, wherein the compound comprises one or more carriers, diluents or excipients.

4. The method of treating obesity in the companion of claim 3, wherein the compound is in a composition comprising phosphate buffered saline and trehalose.

5. The method of treating obesity in the companion animal of claim 1, wherein the compound is administered subcutaneously.

Description

EXAMPLE 1

(1) The polypeptide of SEQ ID NO:1 is obtained essentially as described in US2012/0149,636. The polypeptide of SEQ ID NO:1 is formulated to 4 mg/mL in 20 mM Tris pH 4.0; 2 M urea; and 50 mM methionine. To this formulated polypeptide, 30K PEG (-Methyl--amimooxyethylcarbamyl, polyoxyethylene, Sunbright ME-300CA, NOF, Japan) is added tat a 10:1 PEG to polypeptide molar ratio. The polypeptide and PEG are mixed gently and conjugation is allowed to proceed for 48-72 hours at 28 C. The PEGylated polypeptide is then purified by routine cation exchange chromatography and formulated to 1 mg/ML in PBS (phosphate buffered saline) 7.4 with 4% trehalose. This process results in a mixture which contains between 1-2% of SEQ ID NO: 1, wherein the first methionine is cleaved off.

(2) In Vitro Biological Data

(3) HEK 293 cells are stably transfected with both the luciferase reporter gene under control of a STAT3 response element and the OB-Rb (leptin receptor), which is expressed on the cell surface. Leptin binds to the leptin receptor and activates STAT3 homodimers and STAT3/STAT1 heterodimers, which interact and bind with the STAT3 sequence response element. This interaction drives expression of the luciferase gene and stimulates cells to produce luminescence. The amount of luminescence is proportional to the activity of the compound. The biological activity is based on the EC50 of a 4-PL sigmoidal curve.

(4) Day One

(5) Cells are seeded into a 96-well plate at 20,000 cells/well and placed in a 37 C., 5% CO2 incubator for 24-30 hours.

(6) Day Two

(7) The starting concentration for wild type (WT) canine leptin and the PEGylated polypeptide of Example 1 is 1200 ng/mL. From that, 400 uL volume is taken from each sample and 4 serial dilutions are made across the rows of the dilution block. First, 300 uL/well of assay medium is added to Col 2-11. Then 100 uL is transferred from Col 1 to Col 2, mixed carefully and slowly (without bubbling) 8-10 times, and then continued transferring 100 uL from Col 2 to Col 3 and repeating until reaching Col 11. Leave Col 12 as un-stimulated control well. Cells are then stimulated with the serial dilutions of the wild type (WT) canine leptin and the PEGylated polypeptide of Example 1. The cells are taken out of the incubator and the medium is carefully aspirated from each well. Then 100 L/well of the serially diluted WT and PEGylated polypeptide is transferred from the deep-well dilution block to the cells in the 96-well assay plates. Duplicate samples are then created for WT and PEGylated polypeptide tested horizontally across the next available row. Plates are incubated in a 37 C., 5% CO.sub.2 incubator for 16-18 hours. The plate format with final concentrations in ng/mL is shown below.

(8) TABLE-US-00003 [ng/mL] 1 2 3 4 5 6 7 8 9 10 11 12 WT 1200 300 75 19 4.68 1.17 0.29 0.073 0.018 0.0046 0.0011 0 cLeptin WT 1200 300 75 19 4.68 1.17 0.29 0.073 0.018 0.0046 0.0011 0 cLeptin Example 1 1200 300 75 19 4.68 1.17 0.29 0.073 0.018 0.0046 0.0011 0 Example 1 1200 300 75 19 4.68 1.17 0.29 0.073 0.018 0.0046 0.0011 0
Day Three

(9) On day three, a substrate is prepared and added to the cells and they are then measured by a Luminometer. Approximately 1-2 hours before the plate is read, one set of Steady-Glo (Promega) reagents is thawed including: one vial of Steady-Glo lyophilized Luciferase Assay Substrate and one vial of Steady-Glo Luciverase Assay buffer per assay plate at room temperature. The entire contents of the luciverase assay buffer (10.5 mL) are pipetted into the lyophilized luciferase substrate. 100 L/well of the reconstituted luciferase substrate is added to each well of the assay plate and the assay plate is covered with aluminum foil and incubated at room temperature for 45-60 minutes on a plate shaker set at 450 to 600 rpm. The plate is then read on a Tecan GENiosPRO plate reader with integration time set to 250 msec. The data is then entered into Excel and an EC50 determination on SigmaPlot Application is carried out and the fold loss activity relative to the WT Leptin and percent relative potency determinations are also carried out. Fold loss activity relative to wild type canine leptin is determined to be the [EC50 of the sample] divided by the [EC50 of wild type leptin]; the % relative potency of leptin proteins to reference standard is calculated by dividing the EC50 of reference standard by the EC50 values of leptin proteins and multiplying by 100. For instance, the % Relative Potency is calculated as the [EC50 of referenced standard] divided by the [EC50 of the sample]100. The following table shows WT and PEGylated polypeptide of Example 1 analyzed using these methods.

(10) TABLE-US-00004 TABLE 1 Exp. 1 Exp. 2 AVG Molecule (EC50) (EC50) EC50 WT Canine 1504 1954 1729 Leptin (Ray Biotech) WT Human 2052 3589 2820 Leptin (Ambrx) Example 1 2183 2679 2431 The results show that the PEGylated polypeptide of Example 1 is active in vitro.
In Vivo Biological Data

(11) The objective of this study is to evaluate the impact of the PEGylated polypeptide of Example 1 upon bodyweight, body composition, and feeding behavior in obese male and female dogs. The PEGylated polypeptide of Example 1 formulated at 5.1 mg/ml in phosphate buffered saline with a pH of 7.4 and 4% (w/v) trehalose.

(12) Eighteen (18) dogs all over one (1) year old are used: nine (9) intact male and nine (9) intact female beagles. The dogs are obese, weighing approximately 12 to 18 kg (26.4 to 39.6 lbs). During the first four weeks of the acclimation period, (or until the desired starting weight is achieved), dogs are fed a laboratory a high fat (approx. 45%) dry food that meets or exceeds the nutritional requirements for maintenance and health. All dogs are fed ad libitum during this portion of the acclimation period to facilitate weight gain. During the last two weeks of the acclimation period and for the remainder of the study, all dogs are fed a commercial normal fat (approx. 12%) dry dog food, in order to reduce endogenous leptin levels and restore leptin sensitivity. Dogs in treatment groups 1 and 2 continue to be provided with food ad libitum throughout the remainder of the study. Animals in treatment group 3 are fed twice per day according to the label instructions for the diet. Animals are allowed ad libitum access to water via bowls or an automatic watering system contained in each cage. No other concomitant medications are administered during the course of the study.

(13) This study is conducted as a randomized block design within each gender. Dogs are randomly assigned to pens. Animals are blocked by baseline (Day 14) bodyweights within each gender. There are three blocks with three males and three blocks with three females. Block one of males consist of the 3 males with lowest bodyweights and block one of females will consist of the 3 females with lowest bodyweights. The second blocks within each gender consist of the 3 males and 3 females with the next lowest bodyweights. The final block within each gender contains the 3 males and 3 females with the highest bodyweights.

(14) The following table displays the treatments, dose regimens, and numbers of animals employed.

(15) TABLE-US-00005 TABLE 2 Treatment Dose Regimen # of Animals 1) Negative Controls treated with SIDx5 Q7 Days 6 animals sterile saline, fed ad libitum (3M/3F) 2) Treated with compound of SIDx5 Q7 Days 6 animals Example 1 - 1 mg/kg, fed ad libitum (3M/3F) 3) Treated with compound of SIDx5 Q7 Days 6 animals Example 1 - 1 mg/kg, fed twice daily (3M/3F)

(16) Animals are divided into two gender groups of nine animals. Within each gender group, animals are ranked lowest to highest based on Day 14 bodyweights. Animals in each gender group are divided into blocks of three animals based on increasing baseline bodyweights. Animals are randomly assigned to pen locations using a randomization table provided by the sponsor.

(17) The control and treated groups are administered via subcutaneous injection on Days 0, 7, 14, 21, 28. Serum samples (approximately 2-3 ml) are obtained prior to enrollment in the study on Day 14 prior to switching the animals to the low fat ration in order to acclimate the dogs to the bleeding procedure and to establish baseline endogenous leptin levels. The following table provides the circulating leptin level at days 14 and 1.

(18) TABLE-US-00006 TABLE 3 Day 14 Day 1 Treatment/Feeding Regimen [Leptin] [Leptin] 1) Negative Controls treated with 9.9 ng/ml 3.4 ng/ml sterile saline, fed ad libitum 2) Treated with compound of 10.7 ng/ml 3.7 ng/ml) Example 1 - 1 mg/kg, fed ad libitum 3) Treated with compound of 7.6 ng/ml 3.8 ng/ml Example 1 - 1 mg/kg, fed twice daily

(19) Animals in all three treatment groups are subjected to DEXA (dual energy x-ray absorptiometry) scans on Day 1, Day 21 and Day 42 (the final study day) to determine the percentages of lean versus adipose tissue.

(20) Daily feed consumption are recorded for animals enrolled in treatment groups 1 and 2 on Days 7 through 42 at approximately the same time each.

(21) Animals in all treatment groups are videotaped 24 hours per day starting on Day 7 and continuing through the end of the study to observe feeding behavior including number of meals per day and average duration of feeding per meal.

(22) The primary endpoint will be change in weekly individual animal bodyweights over the course of the study, and these will be summarized and compared to treatment groups. Secondary endpoints will be:

(23) 1) Body Composition: Results of individual animal DEXA scans including the percentages of lean and adipose tissue will be summarized over the course of the study; and

(24) 2) Feed Consumption/Feeding Behavior: Average daily feed consumption for individual animals in Treatments 1 and 2 will be summarized for each animal.

(25) Videotapes recording the feeding behavior of individual animals will be observed to determine the number of meals and duration of meals each animal consumes prior to the initiation of treatment and following the initiation of treatment.

(26) Results of the impact on body weights are provided in the following table.

(27) TABLE-US-00007 TABLE 4 Group Weight Loss (kg) Group 1 - control 0.2 (1.3%) Group 2 - treated 2.2 (16.1%).sup.A Group 3 - treated 2.1 (14.9%).sup.B .sup.Ap = 0.001; .sup.Bp = 0.002.
These results indicate that the groups treated with compound of Example 1 lost more weight as compared to the control group.

(28) The impact on body composition is provided in the following table

(29) TABLE-US-00008 TABLE 5 DEXA DEXA Group BCS Fat Lean Group 1 - 6.8/6.8 8.1% +3.1% control Group 2 - 6.8/5.0.sup.A 40.2%.sup.C 3.7% treated Group 3 - 7.2/5.5.sup.B 36.6.sup.D 1.7% treated .sup.Ap = 0.006; .sup.Bp = 0.049; .sup.Cp = 0.022; .sup.Dp = 0.038.
These results indicate that the groups treated with compound of Example 1 had a reduction in body condition scores associated with a preferential decrease in the percentage of adipose tissue and no significant impact on the percentage of tissue as compared to the control group.

(30) The impact on feed consumption and behavior is provided in the following table.

(31) TABLE-US-00009 TABLE 6 Feed Avg. Consumed Feeding Group (gm) Avg. Meals/Day Time/Day Group 1 257.6 5/4 24/20 Group 2 157.4 5/4 23/15 Group 3 184.9 5/4 32/19
These results indicate that the groups treated with compound of Example consumed less food as compared to the control group and the reduction in food intake was associated with reduced time spent eating as opposed to the number of meals consumed per day.