Long-acting growth hormone and methods of producing same

Abstract

Use of a growth hormone protein and polynucleotides encoding the same comprising an amino-terminal carboxy-terminal peptide (CTP) of chorionic gonadotrophin and two carboxy-terminal chorionic gonadotrophin CTPs attached to the growth hormone in methods of inducing weight loss or body fat reduction, methods of increasing insulin-like growth factor (IGF-1) levels, and methods of reducing the dosing frequency of a growth hormone in a human subject are disclosed. Pharmaceutical compositions comprising the growth hormone and polynucleotides encoding the growth hormone of the invention and methods of using same are also disclosed.

Description

[0001] References to related applications

[0002] This application claims priority to U.S. Application Serial No. US 13 / 195,931, filed August 2, 2011, which is a continuation-in-part of U.S. Patent Application Serial No. US 12 / 509,188, filed July 24, 2009, which US 12 / 509,188 is a continuation of U.S. Patent Application Serial No. US12 / 476,916, filed June 2, 2009, which is a continuation of U.S. Patent Application Serial No. US12 / 401,746, filed March 11, 2009, which US12 / 401,746 is a continuation of US Patent Application Serial No. US 11 / 700,911, filed February 1, 2007, which claims the benefit of US Provisional Application Serial No. US 60 / 764,761, filed February 3, 2006. All of these applications are hereby incorporated by reference in their entirety. technical field

[0003] The present invention discloses growth hormone protein and the polynucleotide encoding the growth hormone protein in the method of inducing weight loss or body fat loss in human subjects, in the meth...

Examples

Embodiment 1

[0212] Generation of hGH constructs

[0213] Materials and Methods

[0214] Four hGH clones (variants of the 20kD protein) were synthesized. The XbaI-NotI fragments containing the hGH sequence from the four variants were ligated into the eukaryotic expression vector pCI-dhfr digested with XbaI-NotI in advance. DNA was prepared from the 4 clones (401-0, 1, 2, 3 and 4). Another partial hGH clone (1-242bp) was also synthesized from the 22kD protein (0606114). Primers were ordered from Sigma-Genosys. The primer sequences used to generate the CTP polypeptide-modified hGH of the present invention are summarized in Table 1 below.

[0215] Table 1

[0216]

[0217] Construction 402-0-p69-l (hGH) (SEQ ID NO:5): hGH is wild-type recombinant human growth hormone (no CTP), which was prepared to be used as a control in the experiments described below.

[0218] Three PCR reactions were performed. The first reaction was performed using primers 25 and 32R and plasmid DNA of 0606114 ...

Embodiment 2

[0230] In vivo biological activity test of the hGH-CTP polypeptide of the present invention

[0231] The following experiments were performed to test the potential long-lasting biological activity of hGH-CTP polypeptides compared to commercial recombinant human GH and hGH.

[0232] Materials and Methods

[0233] Female hypophysectomized rats (60-100 g) received weekly subcutaneous injections of 21.7 μg hGH-CTP polypeptide or 5 μg subcutaneous control commercial rhGH once daily.

[0234] All animals were weighed prior to treatment, 24 hours after the first injection and every other day thereafter until the end of the study on day 21. Each point represents the group's average weight gain rate ((body weight on day 0 - body weight on the last day) / body weight on day 0). Mean body weight gain was normalized to the body weight of once daily injections of commercial hGH. The treatment schedule is summarized in Table 2.

[0235] Table 2

[0236]

[0237] result

[0238] The r...

Embodiment 3

[0242] Pharmacokinetic study of CTP-modified GH

[0243] Single-dose pharmacokinetic studies were performed in Sprague-Dawley rats. All animal experiments were performed in accordance with the Animal Welfare Act, Guidelines for the Care and Use of Laboratory Animals, and under the supervision and approval of the Laboratory Animal Care and Use Committee of Modigene, Biotechnology General Ltd. Rats were housed in a room with a 12-h light / dark cycle, single or two per cage. Optionally provide water (municipal supply) and non-certified rodent chow.

[0244] To compare the pharmacokinetics of CTP-hGH-CTP-CTP and GH in rats, four groups of Sprague-Dawley rats (270–290 g), with 3 to 6 male rats in each group, were used. Rats were randomly assigned to four treatment groups (see Table 3). A single subcutaneous or intravenous injection of GH (50 μg / kg IV or SC) or CTP-hGH-CTP-CTP (108 μg / kg IV or SC) was administered to rats. In addition to pre-dose samples collected under isofluran...

[0001] References to related applications

[0002] This application claims priority to U.S. Application Serial No. US 13 / 195,931, filed August 2, 2011, which is a continuation-in-part of U.S. Patent Application Serial No. US 12 / 509,188, filed July 24, 2009, which US 12 / 509,188 is a continuation of U.S. Patent Application Serial No. US12 / 476,916, filed June 2, 2009, which is a continuation of U.S. Patent Application Serial No. US12 / 401,746, filed March 11, 2009, which US12 / 401,746 is a continuation of US Patent Application Serial No. US 11 / 700,911, filed February 1, 2007, which claims the benefit of US Provisional Application Serial No. US 60 / 764,761, filed February 3, 2006. All of these applications are hereby incorporated by reference in their entirety. technical field

[0003] The present invention discloses growth hormone protein and the polynucleotide encoding the growth hormone protein in the method of inducing weight loss or body fat loss in human subjects, in the meth...

Examples

Embodiment 1

[0212] Generation of hGH constructs

[0213] Materials and Methods

[0214] Four hGH clones (variants of the 20kD protein) were synthesized. The XbaI-NotI fragments containing the hGH sequence from the four variants were ligated into the eukaryotic expression vector pCI-dhfr digested with XbaI-NotI in advance. DNA was prepared from the 4 clones (401-0, 1, 2, 3 and 4). Another partial hGH clone (1-242bp) was also synthesized from the 22kD protein (0606114). Primers were ordered from Sigma-Genosys. The primer sequences used to generate the CTP polypeptide-modified hGH of the present invention are summarized in Table 1 below.

[0215] Table 1

[0216]

[0217] Construction 402-0-p69-l (hGH) (SEQ ID NO:5): hGH is wild-type recombinant human growth hormone (no CTP), which was prepared to be used as a control in the experiments described below.

[0218] Three PCR reactions were performed. The first reaction was performed using primers 25 and 32R and plasmid DNA of 0606114 ...

Embodiment 2

[0230] In vivo biological activity test of the hGH-CTP polypeptide of the present invention

[0231] The following experiments were performed to test the potential long-lasting biological activity of hGH-CTP polypeptides compared to commercial recombinant human GH and hGH.

[0232] Materials and Methods

[0233] Female hypophysectomized rats (60-100 g) received weekly subcutaneous injections of 21.7 μg hGH-CTP polypeptide or 5 μg subcutaneous control commercial rhGH once daily.

[0234] All animals were weighed prior to treatment, 24 hours after the first injection and every other day thereafter until the end of the study on day 21. Each point represents the group's average weight gain rate ((body weight on day 0 - body weight on the last day) / body weight on day 0). Mean body weight gain was normalized to the body weight of once daily injections of commercial hGH. The treatment schedule is summarized in Table 2.

[0235] Table 2

[0236]

[0237] result

[0238] The r...

Embodiment 3

[0242] Pharmacokinetic study of CTP-modified GH

[0243] Single-dose pharmacokinetic studies were performed in Sprague-Dawley rats. All animal experiments were performed in accordance with the Animal Welfare Act, Guidelines for the Care and Use of Laboratory Animals, and under the supervision and approval of the Laboratory Animal Care and Use Committee of Modigene, Biotechnology General Ltd. Rats were housed in a room with a 12-h light / dark cycle, single or two per cage. Optionally provide water (municipal supply) and non-certified rodent chow.

[0244] To compare the pharmacokinetics of CTP-hGH-CTP-CTP and GH in rats, four groups of Sprague-Dawley rats (270–290 g), with 3 to 6 male rats in each group, were used. Rats were randomly assigned to four treatment groups (see Table 3). A single subcutaneous or intravenous injection of GH (50 μg / kg IV or SC) or CTP-hGH-CTP-CTP (108 μg / kg IV or SC) was administered to rats. In addition to pre-dose samples collected under isofluran...

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