Nils Langeland Johansen

Transglutaminase-mediated methods for site-selective modification of human growth hormone†

Two methods for the site‐selective modification of native human growth hormone (hGH) using microbial transglutaminase were developed. In the first method, 1,3‐bisaminoxypropane was attached to hGH, providing a direct incorporation of reactive aminoxy groups for further modification. The reaction was shown to be selective for Gln141, with minor modification at Gln40. In the second method, modified glutamine substrates were developed for attachment to Lys145 in hGH. A series of glutamine‐substrates were screened, and it was shown that microbial transglutaminase was selective towards substitutions on the glutamine core structure. Products from both methods could be transformed to site selectively mono‐PEGylated hGH‐derivatives in good isolated yield.

Aminomethylthiophene-2-carboxylic acids as dipeptide mimetic in new growth hormone secretagogues

Abstract 3-Aminomethylbenzoic acid is a well established dipeptide mimetic. Herein, aminomethylthiophene-2-carboxylic acids 1) have been synthesized as analogues of 3-aminomethylbenzoic acid. Their use as a dipeptide-mimetic at the N -terminal of novel growth hormone secretagogues is described.

Demonstration of the strength of focused combinatorial libraries in SAR optimisation of growth hormone secretagogues

Abstract Aseries of 96 growth hormone secretagogues, derived from ipamorelin are described. The compounds are prepared as a 6 × 4 × 4 member library on solid support using a PAL resin. The compounds are all acylated dipeptides, based on two aromatic amino acids and a free amino N-terminal. All compounds are characterised by HPLC, LC-MS and their ability to release GH in a pituitary cell based assay. The most potent compounds show EC50 values at 1 nM and are full agonists. We demonstrate the strength of focused combinatorial libraries and confirm the pitfall in broad SAR exploration by giving examples where selected fragments obviously show poor receptor interaction except in very defined structural arrangements.

Synthesis and in vitro characterization of new growth hormone secretagogues derived from ipamorelin with dipeptidomimetic N-terminals

The structural requirements for N-terminal features for the minimal structure of growth hormone secretagogues derived from ipamorelin are investigated. It is found, that incorporation of nonpolar peptidomimetic amino acids at the N-terminal can replace the Aib-His moiety and lead to compounds with high in vitro potency with respect to their growth hormone secretagogue properties. New unnatural amino acids with double bonds, ether-linkages, and 1,3-phenylene-moieties in the backbone proved to be valuable dipeptidomimetics. Using them, growth hormone secretagogues with high potencies were obtained.

Solid phase synthesis of ψ[CH(CN)NH] pseudopeptides. Application to the synthesis of analogues of neurotensin [NT(8–13)].

Abstract The introduction of the cyanomethyleneamino [CH(CN)NH] group, a new type of peptide bond surrogate, under solid phase peptide synthesis conditions has been studied using both Boc and Fmoc strategies. The pseudohexapeptides H-Arg-Pro-Tyrψ[CH(CN)NH]Ile-Leu-OH ( 1 ) and H-Arg-Arg-Pro-Tyr-Ileψ[CH(CN)NH]Leu-OH ( 2 ), analogues of the C -terminal hexapeptide of neurotensin [NT(8–13)], were successfully obtained via Fmoc synthesis.

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT8−13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro10-Tyr11 fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III β-turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT8−13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala11]-NT8−13 and only five- to ninefold lower than that of the corresponding analogue, [Phe11]NT8−13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT8−13 analogues and [Phe11]NT8−13 or NT8−13, it is not clear whether the β-turn around Pro10-AA11 (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro10-Tyr11 dipeptide in NT8−13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.

Nonclinical pharmacokinetic and pharmacodynamic characterisation of somapacitan: A reversible non-covalent albumin-binding growth hormone

OBJECTIVE Somapacitan is an albumin-binding growth hormone derivative intended for once weekly administration, currently in clinical development for treatment of adult as well as juvenile GH deficiency. Nonclinical in vivo pharmacological characterisation of somapacitan was performed to support the clinical trials. Here we present the pharmacokinetic and pharmacodynamic effects of somapacitan in rats, minipigs, and cynomolgus monkeys. METHODS Pharmacokinetic studies investigating exposure, absorption, clearance, and bioavailability after single intravenous (i.v.) and subcutaneous (s.c.) administration were performed in all species. A dose-response study with five dose levels and a multiple dose pharmacodynamic study with four once weekly doses was performed in hypophysectomised rats to evaluate the effect of somapacitan on growth and IGF-I production. RESULTS Pharmacokinetic profiles indicated first order absorption from the subcutaneous tissue after s.c. injections for somapacitan in all three species. Apparent terminal half-lives were 5-6h in rats, 10-12h in minipigs, and 17-20h in monkeys. Somapacitan induced a dose-dependent growth in hypophysectomised rats (p<0.001) and an increase in plasma IGF-I levels in rats (p<0.01), minipigs (p<0.01), and cynomolgus monkeys (p<0.05) after single dose administration. Multiple once weekly dosing of somapacitan in hypophysectomised rats induced a step-wise increase in body weight with an initial linear phase the first 3-4days in each dosing interval (p<0.001). CONCLUSION The nonclinical pharmacokinetic and pharmacodynamic studies of somapacitan showed similar pharmacokinetic properties, with no absorption-limited elimination, increased clearance and increased and sustained levels of IGF-I in plasma for up to 10days after a single dose administration in all three species. Somapacitan induced a dose-dependent increase in body weight and IGF-I levels in hypophysectomised rats. Multiple dosing of somapacitan in hypophysectomised rats suggested a linear growth for the first 3-4days in each weekly dosing interval, whereas daily hGH dosing showed linear growth for approximately two weeks before reaching a plateau level.

Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.

GP3-6: Mixed effects PK/PD modelling of NNC0195-0092 - a noncovalent albumin binding growth hormone derivative in the hypophysectomised rat

Introduction: The hypophysectomised rat is used during preclinical development to evaluate the pharmacokinetic and pharmacodynamic (PKPD) properties of new GH analogues . The animal model resembles the human GH deficiency with lack of GH secretion, low levels of serum IGF-I and growth retardation . Non-linear mixed effects modelling of the PKPD relationship may improve the mechanistic understanding of action, and form the basis for translation of pre-clinical parameters into meaningful and accurate human predictions for early clinical development . A mechanistic mixed-effects PKPD model for NNC0195-0092 a non-covalent albumin binding growth hormone derivative has been developed in hypophysectomised Sprague Dawley rats . Methods: Animal studies with single and multiple dosing of NNC0195-0092 was used to establish the PKPD relationship between NNC0195-0092 and insulin-like growth factor I (IGF-I) Non-linear mixed effects modelling software (NONMEM) was used to model the simultaneous relationship between NNC01950092, IGF-I and bodyweight gain . Derived parameters was scaled directly from model values using the allometric approach with exponents fixed to conventional values . Results: A final PKPD model was established, that described the PK of NNC0195-0092 as a two compartmental model with both linear and non-linear elimination terms, transit compartment absorption . The induction of IGF-I was modelled using an indirect response model with stimulation of kin with an Emax relationship to rhGH dose . Direct scaling form the PKPD model provided good predictions of the human PKPD except for subcutaneous bioavailability were the approaches failed . Conclusion: A PKPD model of pre-clinical animal data was developed . The model provides a quantitative way of comparing results from testing of new GH analogues in the hypophysectomised rat model to current treatment . Furthermore, direct scaling of parameters matched or improved results obtained when using multispecies scaling to predict human parameters .

Design, synthesis and biological activity of cyclic GHRP-6 analogues

Growth Hormone (GH) secretion is normally stimulated by GHRH. In 1984 C.Y. Bowers and F.A. Momany synthesized the hexa-peptide Growth Hormone Releasing Peptide (GHRP-6) and discovered its ability to stimulate GH release in various species [1]. With the aim to identify the bioactive conformation of GHRP-6, a series of constrained monocyclic analogs were prepared. The cyclizations involved the termini and/or the side chains and, in some of the analogs, a reduced peptide bond was also introduced.