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Oral growth hormone enhancer MK-677 (ibutamoren)
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<blockquote data-quote="BigTex" data-source="post: 239824" data-attributes="member: 43589"><p>This shoudl explain the difference in natural GLP-1 foundint he gut and GLP-1 analogues</p><p></p><p>Semaglutide is a human GLP-1 analogue in clinical development for the treatment of T2D. It shares 94% structural homology with native human GLP-1, with three important modifications: amino-acid substitutions at position 8 (alanine to alpha-aminoisobutyric acid) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a <a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/glutamic-acid" target="_blank">glutamic acid</a> moiety, and a C-18 fatty di-acid side chain (<a href="https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075" target="_blank">Lau et al., 2015</a>). The fatty di-acid side chain and spacer mediate strong binding with albumin, while the amino-acid substitution at position 34 limits the options for acylation to the one remaining lysine in the sequence; the substitution at position 8 reduces the susceptibility of semaglutide to degradation by DPP-4 (<a href="https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075" target="_blank">Lau et al., 2015</a>). Together, these modifications prolong the t1/2 of semaglutide; a previous pharmacokinetic (PK) trial with semaglutide showed the geometric means (coefficient of variation in %) of t1/2 was 165 (14.1) h (approximately 1 week). Additionally, the dose dependent increase of the Ctrough values following 4 weeks of treatment with semaglutide 0.25 mg, 0.5 mg and 1.0 mg was 4.4 (31.5), 11.7 (20.2) and 21.2 (19.7) nmol/L, respectively (<a href="https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0070" target="_blank">Kapitza et al., 2015</a>).</p><p></p><p>[ATTACH=full]27153[/ATTACH]</p><p>Changes to semaglutide compared with native human GLP-1 are: amino-acid substitutions at position 8 (alanine to alpha-aminoisobutyric acid) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with two ADO moieties followed by a glutamic acid moiety (the ‘spacer’) and a C-18 fatty di-acid side chain (<a href="https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075" target="_blank">Lau et al., 2015</a>). ADO, 8-amino-3,6-dioxaoctanoic acid.</p><p></p><h4>2.1.1. Synthetic procedure for the radioactive building block [3H]-NNC0113-0857</h4><p>The starting material, NNC0113-3747 (6.6 mg, 6.5 μmol), was dissolved in a solution of N-methyl-2-pyrrolidone (NMP) (1.0 mL) and <a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/trifluoroacetic-acid" target="_blank">trifluoroacetic acid</a> (TFA) (5 μL) before 10 wt% Pd/C (6.5 mg) was added. The mixture was subjected to tritium gas (6.9 Ci) and stirred at room temperature. After 4 h, Pd/C was filtered out and the residue co-evaporated (3 × 1.0 mL) with a 1000:1 solution of EtOH/TFA. The residue was dissolved with NMP (1.0 mL) and the radioactive content was determined (180 mCi/mL, 1.6 mL). High-performance liquid chromatography (HPLC) analysis showed that [3H]-NNC0113-0857 had radiochemical purity of 61%.</p><p></p><h4>2.1.2. Synthetic procedure for [3H]-semaglutide</h4><p>The peptide backbone (20.4 mg, 6.0 μmol), was dissolved in H2O (5.0 mL) and the pH adjusted to 11 via the addition of NEt3. The solution of [3H]-NNC0113-0857 in NMP (180 mCi/mL, 1.6 mL) was slowly added over 15 min. After 2 h, the reaction was quenched and neutralised to pH 7 by addition of 1 M CH3COOH (120 μL). The reaction mixture was purified by HPLC (column: Luna® reversed phase C18; A-eluent [0.1 M Tris-HCl, 0.05 M·H3PO4, H2O]/EtOH 8:2, pH 7.4; B-eluent [0.1 M Tris-HCI, 0.05 M·H3PO4, H2O]/EtOH 55:45, pH 7.4) and yielded a [3H]-semaglutide solution of 98% purity. [3H]-semaglutide was desalted on a C18 Sep-Pak® filter and eluted with H2O/EtOH (3:7) into a solution with a radioactive content of 16.7 mCi/mL. The solution was concentrated to dryness on a rotary evaporator and then reconstituted in the following vehicle/API solution of semaglutide (1.34 mg/mL, 1.0 mg/mL or 0 mg/mL), <a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/propylene-glycol" target="_blank">propylene glycol</a> (14 mg/mL), Na2HPO4·2H2O (1.42 mg/mL), phenol (5.5 mg/mL), pH 7.4. HPLC analysis of formulated [3H]-semaglutide gave a radiochemical purity of > 98%.</p><p></p><h4>2.1.3. Stability of [3H]-semaglutide</h4><p>[3H]-semaglutide stability in vehicle was assessed over a 15-day period at 5 °C and 25 °C and confirmed. Radiochemical purity was determined by HPLC. Prestudy stability tests revealed that the tritium tracer showed a satisfactory stability. The amount of tritiated water formed after 15 days’ storage at 25 °C was low (< 0.1%), as was the amount of impurities (~ 2% at 5 °C and ~ 5% at 25 °C).</p><p></p><h4>2.1.4. Stability of semaglutide in human material</h4><p>Stability of the tracer was also assessed in plasma and urine and showed that the [3H]-semaglutide tracer was stable in human blood, plasma and urine for up to at least 4 h at room temperature and up to at least 48 h at nominal + 4, − 20 and − 70 °C. The stability of the [3H]-semaglutide radiotracer was, therefore, considered to be adequate for conducting metabolism studies.</p><p></p><p>[URL unfurl="true"]https://www.sciencedirect.com/science/article/pii/S0928098717301537[/URL]</p></blockquote><p></p>
[QUOTE="BigTex, post: 239824, member: 43589"] This shoudl explain the difference in natural GLP-1 foundint he gut and GLP-1 analogues Semaglutide is a human GLP-1 analogue in clinical development for the treatment of T2D. It shares 94% structural homology with native human GLP-1, with three important modifications: amino-acid substitutions at position 8 (alanine to alpha-aminoisobutyric acid) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with a spacer consisting of two 8-amino-3,6-dioxaoctanoic acid (ADO) moieties, a [URL='https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/glutamic-acid']glutamic acid[/URL] moiety, and a C-18 fatty di-acid side chain ([URL='https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075']Lau et al., 2015[/URL]). The fatty di-acid side chain and spacer mediate strong binding with albumin, while the amino-acid substitution at position 34 limits the options for acylation to the one remaining lysine in the sequence; the substitution at position 8 reduces the susceptibility of semaglutide to degradation by DPP-4 ([URL='https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075']Lau et al., 2015[/URL]). Together, these modifications prolong the t1/2 of semaglutide; a previous pharmacokinetic (PK) trial with semaglutide showed the geometric means (coefficient of variation in %) of t1/2 was 165 (14.1) h (approximately 1 week). Additionally, the dose dependent increase of the Ctrough values following 4 weeks of treatment with semaglutide 0.25 mg, 0.5 mg and 1.0 mg was 4.4 (31.5), 11.7 (20.2) and 21.2 (19.7) nmol/L, respectively ([URL='https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0070']Kapitza et al., 2015[/URL]). [ATTACH type="full"]27153[/ATTACH] Changes to semaglutide compared with native human GLP-1 are: amino-acid substitutions at position 8 (alanine to alpha-aminoisobutyric acid) and position 34 (lysine to arginine), and acylation of the lysine in position 26 with two ADO moieties followed by a glutamic acid moiety (the ‘spacer’) and a C-18 fatty di-acid side chain ([URL='https://www.sciencedirect.com/science/article/pii/S0928098717301537#bb0075']Lau et al., 2015[/URL]). ADO, 8-amino-3,6-dioxaoctanoic acid. [HEADING=3]2.1.1. Synthetic procedure for the radioactive building block [3H]-NNC0113-0857[/HEADING] The starting material, NNC0113-3747 (6.6 mg, 6.5 μmol), was dissolved in a solution of N-methyl-2-pyrrolidone (NMP) (1.0 mL) and [URL='https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/trifluoroacetic-acid']trifluoroacetic acid[/URL] (TFA) (5 μL) before 10 wt% Pd/C (6.5 mg) was added. The mixture was subjected to tritium gas (6.9 Ci) and stirred at room temperature. After 4 h, Pd/C was filtered out and the residue co-evaporated (3 × 1.0 mL) with a 1000:1 solution of EtOH/TFA. The residue was dissolved with NMP (1.0 mL) and the radioactive content was determined (180 mCi/mL, 1.6 mL). High-performance liquid chromatography (HPLC) analysis showed that [3H]-NNC0113-0857 had radiochemical purity of 61%. [HEADING=3]2.1.2. Synthetic procedure for [3H]-semaglutide[/HEADING] The peptide backbone (20.4 mg, 6.0 μmol), was dissolved in H2O (5.0 mL) and the pH adjusted to 11 via the addition of NEt3. The solution of [3H]-NNC0113-0857 in NMP (180 mCi/mL, 1.6 mL) was slowly added over 15 min. After 2 h, the reaction was quenched and neutralised to pH 7 by addition of 1 M CH3COOH (120 μL). The reaction mixture was purified by HPLC (column: Luna® reversed phase C18; A-eluent [0.1 M Tris-HCl, 0.05 M·H3PO4, H2O]/EtOH 8:2, pH 7.4; B-eluent [0.1 M Tris-HCI, 0.05 M·H3PO4, H2O]/EtOH 55:45, pH 7.4) and yielded a [3H]-semaglutide solution of 98% purity. [3H]-semaglutide was desalted on a C18 Sep-Pak® filter and eluted with H2O/EtOH (3:7) into a solution with a radioactive content of 16.7 mCi/mL. The solution was concentrated to dryness on a rotary evaporator and then reconstituted in the following vehicle/API solution of semaglutide (1.34 mg/mL, 1.0 mg/mL or 0 mg/mL), [URL='https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/propylene-glycol']propylene glycol[/URL] (14 mg/mL), Na2HPO4·2H2O (1.42 mg/mL), phenol (5.5 mg/mL), pH 7.4. HPLC analysis of formulated [3H]-semaglutide gave a radiochemical purity of > 98%. [HEADING=3]2.1.3. Stability of [3H]-semaglutide[/HEADING] [3H]-semaglutide stability in vehicle was assessed over a 15-day period at 5 °C and 25 °C and confirmed. Radiochemical purity was determined by HPLC. Prestudy stability tests revealed that the tritium tracer showed a satisfactory stability. The amount of tritiated water formed after 15 days’ storage at 25 °C was low (< 0.1%), as was the amount of impurities (~ 2% at 5 °C and ~ 5% at 25 °C). [HEADING=3]2.1.4. Stability of semaglutide in human material[/HEADING] Stability of the tracer was also assessed in plasma and urine and showed that the [3H]-semaglutide tracer was stable in human blood, plasma and urine for up to at least 4 h at room temperature and up to at least 48 h at nominal + 4, − 20 and − 70 °C. The stability of the [3H]-semaglutide radiotracer was, therefore, considered to be adequate for conducting metabolism studies. [URL unfurl="true"]https://www.sciencedirect.com/science/article/pii/S0928098717301537[/URL] [/QUOTE]
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Oral growth hormone enhancer MK-677 (ibutamoren)
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