Half-life extension: how chemists make a peptide last longer in blood

Why a small peptide is cleared in minutes

Review Two processes shorten the life of a peptide in blood: proteolytic enzymes degrade it, and the kidneys clear it.¹¹

Enzymes

Cell study Human plasma degraded GLP-1(7-36)amide with a half-life of about 20 minutes at body temperature, and the product was the peptide without its first two residues.¹² Inhibitors of DPP-IV, also written DPP-4, prevented its formation completely.¹²

Cell study Mentlein and colleagues described DPP-IV as a specialized enzyme that removes a dipeptide only when the second residue from the N-terminus is proline or alanine.² The purified enzyme cut GLP-1(7-36)amide, GIP and GHRH(1-29)amide, each of which has alanine in that position.²

Human study Frohman and colleagues followed GHRH(1-44)-NH2 by HPLC: in plasma in vitro the intact hormone had a half-life of 17 minutes, and in normal subjects it was 6.8 minutes, with the shortened product detected within one minute.¹ That product, GHRH(3-44)-NH2, had less than one-thousandth of the hormone’s biological activity.¹

The kidney

Review A review of GHRH analogues puts the half-life of GHRH(1-29) at about 10 to 20 minutes in humans, and attributes it mostly to ultrafiltration by the kidney and to enzymatic degradation at the N-terminus.¹³

Strategy 1: change what the enzyme recognizes

Cell study In plasma, GHRH analogues with a D-amino acid at position 1 or 2 were not cleaved by DPP-IV.³ The same study found a second, lesser route: trypsin-like cleavage between residues 11 and 12.³

Semaglutide carries the non-coded amino acid Aib at position 8, a change its label says stabilizes it against DPP-4.⁷¹⁴ The article on peptide nomenclature and modifications explains how such changes are written.

Animal study Tesamorelin, development code TH9507, takes another route: a trans-3-hexenoyl group on the first residue of GHRH(1-44).⁴¹⁵ Its developers reported that it resisted DPP-IV, broke down more slowly than natural GHRH in rat, dog and human plasma, and had an apparent elimination half-life of 21 to 45 minutes in dogs.⁴

Human study The FDA label for tesamorelin gives a mean elimination half-life of 8 minutes in healthy subjects.⁵ It and the 6.8 minutes reported for the natural hormone come from different studies and methods.¹

Strategy 2: bind albumin with a fatty acid

Semaglutide’s label explains the principle: binding to albumin, a plasma protein, lowers clearance by the kidney and protects the peptide from metabolic degradation.⁷

Cell study A 2000 paper from Novo Nordisk described GLP-1 derivatives carrying fatty acids, made to prolong their action through binding to serum albumin.¹⁶ Every compound with a chain of twelve carbons or more was much longer-acting than GLP-1.¹⁶ Many kept a potency at the cloned human GLP-1 receptor similar to that of GLP-1, but the longer the fatty acid the more potency was lost, and two fatty acids caused a considerable loss.¹⁶

Animal study The designers of semaglutide set out to raise albumin affinity further, and reported that the fatty acid and the chemistry linking it to the peptide were the key features.¹⁴ Its affinity for the GLP-1 receptor was three-fold lower than liraglutide’s, its albumin affinity was higher, and its plasma half-life in mini-pigs was 46.1 hours.¹⁴

Human study Liraglutide’s label describes a C16 fatty acid joined through a glutamic acid spacer to the lysine at position 26, more than 98 per cent binding to plasma protein, and an elimination half-life of about 13 hours.⁶ The same label gives GLP-1(7-37) a half-life of 1.5 to 2 minutes.⁶ The labels put the half-life of semaglutide, with a C18 fatty diacid, at about one week, and that of tirzepatide, with a C20 fatty diacid, at about five days.⁷¹⁷

The article on incretin receptor agonists compares those molecules.

Strategy 3: conjugate to albumin, the drug affinity complex

The next design uses a reactive group to conjugate the peptide to albumin itself.

Animal study Jetté and colleagues at ConjuChem made maleimide derivatives of GHRH(1-29), designed to react with the free thiol on cysteine 34 of albumin, and named the best of them CJC-1295.⁸ Conjugates prepared with human albumin outside the body were more stable against DPP-IV in vitro and were active in a growth hormone secretion assay on cultured rat pituitary cells.⁸ In rats, CJC-1295 was present in plasma beyond 72 hours, and a blot of plasma showed it on the albumin band after 15 minutes.⁸

Animal study A 2003 paper with several of the same authors used the name drug affinity complex (DAC) for CJC-1131, a GLP-1 compound that conjugates to albumin in vivo, studied in mice.¹⁸

Human study In two randomized, placebo-controlled, double-blind trials in healthy adults aged 21 to 61, the estimated half-life of CJC-1295 was 5.8 to 8.1 days.⁹

In the 2005 paper the name CJC-1295 means the molecule with the maleimide group.⁸ The catalogue also lists a blend labelled “no DAC”. Nothing measured on the long-acting compound carries over to a peptide without that group.

Strategy 4: add a polymer

Review Pegylation attaches chains of polyethylene glycol (PEG), which raise molecular mass and shield the peptide from proteolytic enzymes.¹¹ A review from Serono of pegylated GHRH(1-29) examined chain size, chain number and attachment site: the conjugates activated the human GHRH receptor with differing potency, and those with a single PEG5000 chain on lysine 12 or lysine 21 kept in-vitro activity similar to the parent’s.¹³

Strategy 5: fuse to an antibody fragment

Animal study Glaesner and colleagues at Eli Lilly made a fusion of GLP-1 with the Fc region of an IgG4 antibody; the protein kept full receptor activity in vitro and had a half-life of 1.5 to 2 days in rats and cynomolgus monkeys.¹⁰

Human study The label for dulaglutide describes a molecule of the same design: two identical chains, each a GLP-1 analogue linked to a modified IgG4 Fc, about 63 kilodaltons, with an elimination half-life of about five days.¹⁹ It is produced in mammalian cell culture, a different route from solid-phase peptide synthesis.¹⁹

The strategies side by side

Human evidence

The human data on this page are pharmacokinetic: they say how long a molecule stays in blood, in healthy volunteers or in the people described on a label.¹⁶⁹ A half-life is not evidence of an effect or of safety. The plasma, rat, dog, mini-pig, mouse and monkey results are not human findings, and where a peptide’s human pharmacokinetics have not been published, nothing here supplies them.

The FDA labels are cited here for structure and pharmacokinetics only, and this article says nothing about any compound’s regulatory status. Products on this site are sold for laboratory research use only, as the research use only policy sets out.