Human growth hormone-releasing factor (hGRF)1-29-albumin bioconjugates activate the GRF receptor on the anterior pituitary in rats: identification of CJC-1295 as a long-lasting GRF analog.

In vivo bioconjugation to the free thiol on Cys34 of serum albumin by a strategically placed reactive group on a bioactive peptide is a useful tool to extend plasma half-life. Three maleimido derivates of human GH-releasing factor (hGRF)(1-29) were synthesized and bioconjugated to human serum albumin ex vivo. All three human serum albumin conjugates showed enhanced in vitro stability against dipeptidylpeptidase-IV and were bioactive in a GH secretion assay in cultured rat anterior pituitary cells. When the maleimido derivatives were individually administered sc to normal male Sprague Dawley rats, an acute secretion of GH was measured in plasma. The best compound, CJC-1295, showed a 4-fold increase in GH area under the curve over a 2-h period compared with hGRF(1-29). CJC-1295, a tetrasubstituted form of hGRF(1-29) with an added N epsilon-3-maleimidopropionamide derivative of lysine at the C terminus, was selected for further pharmacokinetic evaluation, where it was found to be present in plasma beyond 72 h. A Western blot analysis of the plasma of a rat injected with CJC-1295 showed the presence of a CJC-1295 immunoreactive species on the band corresponding to serum albumin, appearing after 15 min and remaining in circulation beyond 24 h. These results led to the identification of CJC-1295 as a stable and active hGRF(1-29) analog with an extended plasma half-life.

PEGylation of growth hormone-releasing hormone (GRF) analogues.

Synthetically produced GRF1-29 (Sermorelin) has an amino acid composition identical to the N-terminal 29 amino acids sequence of the natural hypothalamic GHRH1-44 (Figure 1). It maintains bioactivity in vitro and is almost equally effective in eliciting secretion of endogenous growth hormone in vivo. The main drawbacks associated with the pharmaceutical use of hGRF1-29 relate to its short half-life in plasma, about 10-20 min in humans, which is caused mostly by renal ultrafiltration and enzymatic degradation at the N terminus. PEGylation has been considered as one valid approach to obtain more stable forms of the peptide, with a longer in vivo half-life and ultimately with increased pharmacodynamic response along the somatotropic axis (endogenous GH, IGF-1 levels). Different PEGylated GRF conjugates were obtained and their bioactivity was tested in vitro and in vivo by monitoring endogenous growth hormone (GH) serum levels after intravenous (i.v.) injection in rats, and intravenous and subcutaneous (s.c.) injection in pigs. It was found that GRF-PEG conjugates are able to bind and activate the human GRF receptor, although with different potency. The effect of PEG molecular weight, number of PEG chains bound and position of PEGylation site on GRF activity were investigated. Mono-PEGylated isomers with a PEG5000 polymer chain linked to Lys 12 or Lys 21 residues, showed high biological activity in vitro, which is similar to that of hGRF1-29, and a higher pharmacodynamic response as compared to unmodified GRF molecule.

The involvement of dipeptidyl peptidase IV in brush-border degradation of GRF(1-29)NH2 by intestinal mucosal cells.

GRF(1-29)NH2 is degraded mainly by dipeptidyl peptidase IV (DPP IV) in plasma, resulting in inactivated GRF(3-29)NH2. To understand whether improving stability of GRF(1-29)NH2 in the plasma will result in enhanced stability in intestinal mucosal cells, stability of GRF(1-29)NH2 and [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)NH2 in rat intestine brush-border membrane and homogenate was examined. [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)NH2, resistant to plasma DPP IV, was much more stable than GRF(1-29)NH2 in enterocytes. Gradient HPLC analysis, mass balance analysis and studies of inhibitor effects revealed that GRF(3-29)NH2 was the major metabolite of GRF(1-29)NH2 due to the action of DPP IV during incubation with brush-border membranes. It is concluded that the design of peptide analogues to resist plasma enzymes dramatically increases stability in intestinal epithelium.

Incorporation of D-Ala2 in growth hormone-releasing hormone-(1-29)-NH2 increases the half-life and decreases metabolic clearance in normal men.

D-Ala2-GHRH-(1-29) has increased binding affinity and exhibits enhanced biological activity in man. It is not known whether changes in the metabolic clearance of this and other GHRH analogs contribute to their increased biological activity. GHRH-(1-29)-NH2 and D-Ala2-GHRH-(1-29)-NH2 were administered by constant iv infusion at a rate of 25 ng/kg.min to 10 normal men. Blood was sampled during the 90-min infusion and for 20 min afterward and assayed for the infused analog. The MCR of the D-Ala2 analog (mean +/- SE) was significantly less (21 +/- 1.2 mL/kg.min) than that of GHRH-(1-29)-NH2 (39.7 +/- 3.9 mL/kg.min; P < 0.001). The disappearance half-time of the D-Ala2 analog was 6.7 +/- 0.5, whereas that of GHRH-(1-29)-NH2 was 4.3 +/- 1.4 min (P < 0.05). These findings demonstrate that the D-Ala2 substitution contributes to the enhancement of biological activity by reducing metabolic clearance.

Pharmacokinetics of growth hormone-releasing hormone(1-29)-NH2 and stimulation of growth hormone secretion in healthy subjects after intravenous or intranasal administration.

The growth hormone-releasing hormone analogue GHRH(1-29)-NH2 was administered intravenously or intranasally to 30 healthy men aged 19-43 years. Intravenous injection of the lowest dose tested, 0.25 microgram/kg body weight, elicited significant release of growth hormone (GH). Maximal release (mean GH peaks of about 90 mU/l) was obtained with a dose of 1-2 micrograms/kg. Although GHRH(1-29)-NH2 was rapidly eliminated after intravenous injection, GH levels were elevated for about 3 hours. Absorption of GHRH(1-29)-NH2 through the nasal mucosa was found to be low, and the bioavailability was only 3-5%. There was a dose-dependent release of GH after intranasal administration of GHRH(1-29)-NH2, with the maximal response obtained with about 50 micrograms/kg; this dose was approximately as potent as 1 microgram/kg injected intravenously. The GH response after repeated intranasal administration of GHRH(1-29)-NH2 was sustained; there was no suppression of GH secretion during the night following a day when GHRH(1-29)-NH2 had been given three times intranasally. Based on these findings and the obvious convenience of intranasal administration compared with injections, it would be justified to test intranasal therapy for treatment of short stature in children with GH deficiency caused by hypothalamic damage.