Long-acting Growth Hormones Produced by Conjugation with Polyethylene Glycol

Abstract

Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the Nhydroxysuccinimide ester of polyethylene glycol-5000 (PEG 5000 ), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC 50 in a cellbased assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG 5000 , and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.The ability of a hormone to elicit a biological effect in vivo depends on many factors including the affinity for its receptor and the rate at which it is cleared from the circulation. Some hormones, like atrial natriuretic peptide, have a very high affinity for their receptor (10 pM) and are cleared very rapidly (t1 ⁄2 ϳ0.5 min) by receptor and protease-mediated events (1). Other hormones, like human growth hormone (hGH), 1 have lower affinity for their receptor (300 pM) but are cleared more slowly (t1 ⁄2 ϳ30 min in rats), primarily via the kidney (2, 3).Understanding the relationships between hormone affinity, clearance, and efficacy is important in optimizing hormone therapy. To study this systematically one would like to vary these parameters and evaluate their relative importance in regulating biopotency. hGH is a good model system in this regard as much is known about its structure and function (for review see Ref. 4). Simple receptor binding (5, 6), cell-based assays (7,8), and growth parameters in rodents (9) can be used to determine biopotency in vitro and in vivo. The properties of proteins such as hGH that are cleared by kidney filtration can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the hormone and thereby slows its clearance (10, for recent review see Ref. 11).Here, we describe a set of hGH derivatives conjugated with increasing numbers of PEG ...

Optimized clinical performance of growth hormone with an expanded genetic code

“…However, while hGH therapy is highly efficacious, it requires daily subcutaneous injection and frequent dose adjustment to minimize adverse effects. Efforts thus far to modify the protein and its formulation have failed to produce a sustained-action hGH with the efficacy, safety, and tolerability of daily subcutaneous injection (5,6). Herein we report the pharmacological properties of a previously unreported hGH molecule biosynthesized with a genetically encoded nonnative amino acid.…”

Development of Pure Prolactin Receptor Antagonists

“…Due to the relatively short half-life (15-20 min) of PRL and GH in vivo (63), analysis of long term effect of the antagonists on prostate gene expression was performed by implanting osmotic mini-pumps in probasin-rPRL animals to ensure delivery of the antagonists (G129R-hPRL or ⌬1-9-G129R-hPRL) at a constant rate over several days. Unfortunately, this approach only allowed an antagonist concentration of ϳ60 -70 ng/ml in serum, which is far from the concentration required to achieve efficient antagonism as shown above.…”

Cross-linked growth hormone dimers have enhanced biological activity

“…Thus the cross-link would be proposed to stabilise the conformation found in native dimers fundamentally altering its structure and altering its bioactivity. Enhancement of GH action has previously been reported when laboratory animals (dwarf mice and dwarf rats) are treated with the hormone complexed to anti-GH monoclonal or polyclonal antibodies (36)(37)(38)(39)(40)(41), the GH binding protein (42) and when covalently linked to polyethylene glycol (43). A number of mechanisms have been suggested to account for this enhancement but as yet the precise mechanism(s) underlying the phenomenon remains unclear.…”

“…The presence of increasing concentrations of EDC in reaction mixtures containing hGH was associated with increasing amounts of 125 I-hGH appearing in the GH dimer region. For all samples the GH dimer peak was found in gel slices [38][39][40][41][42][43]. Counts in these sections (minus background) were summed and expressed as a percentage of counts loaded onto the gel in order to calculate the percentage cross-linking of 125 I-hGH in each cross-linking reaction; these values are given in Table 1.…”