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.

A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.

Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.

Structure-based design of selective and potent inhibitors of protein-tyrosine phosphatase beta.

Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.

A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta).

Islet-cell antigen 512 (IA-2) and phogrin (IA-2beta) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent studies indicate that IA-2 may be involved in granule trafficking and exocytosis. To further understand their function, we have embarked upon developing low-molecular-mass inhibitors of IA-2 and IA-2beta. Previously, we have shown that a general PTP inhibitor, 2-(oxalylamino)benzoic acid (OBA), can be developed into highly selective and potent inhibitors of PTP1B. However, since wild-type IA-2 and IA-2beta lack conventional PTP activity, a novel strategy was designed whereby catalytically active species were generated by 'back-mutating' key non-consensus catalytic region residues to those of PTP1B. These mutants were then used as tools with which to test the potency and selectivity of OBA and a variety of its derivatives. Catalytically competent IA-2 and IA-2beta species were generated by 'back-mutation' of only three key residues (equivalent to Tyr(46), Asp(181) and Ala(217) using the human PTP1B numbering) to those of PTP1B. Importantly, enzyme kinetic analyses indicated that the overall fold of both mutant and wild-type IA-2 and IA-2beta was similar to that of classic PTPs. In particular, one derivative of OBA, namely 7-(1,1-dioxo-1 H -benzo[ d ]isothiazol-3-yloxymethyl)-2-(oxalylamino)-4,7-dihydro-5 H -thieno[2,3- c ]pyran-3 -carboxylic acid ('Compound 6 ' shown in the main paper), which inhibited IA-2beta((S762Y/Y898P/D933A)) (IA-2beta in which Ser(762) has been mutated to tyrosine, Tyr(898) to proline, and Asp(933) to alanine) with a K (i) value of approximately 8 microM, appeared ideal for future lead optimization. Thus molecular modelling of this classical, competitive inhibitor in the catalytic site of wild-type IA-2beta identified two residues (Ser(762) and Asp(933)) that offer the possibility for unique interaction with an appropriately modified 'Compound 6 '. Such a compound has the potential to be a highly selective and potent active-site inhibitor of wild-type IA-2beta.

Discovery and SAR of a novel selective and orally bioavailable nonpeptide classical competitive inhibitor class of protein-tyrosine phosphatase 1B.

Reversible phosphorylation and dephosphorylation of key proteins on tyrosine residues are important parts of intracellular signaling triggered by hormones and other agents. Recent knock-out studies in mice have identified PTP1B as a potential target for the treatment of diabetes and obesity. As a consequence, a number of academic and industrial groups are aggressively pursuing the development of selective PTP1B inhibitors. In addition, other protein-tyrosine phosphatases (PTPs) appear to be critically involved in major diseases such as cancer and autoimmunity. Given the diversity of PTPs and their potential as drug targets in different diseases, we have taken a broad approach to develop active site-directed selective inhibitors of specific members of this family of enzymes. Using a high throughput screening, we have previously identified 2-(oxalylamino)benzoic acid 3a as a relatively weak but classical competitive inhibitor of several PTPs.(4) On the basis of our early studies, indicating that 3a might be used as a starting point for the synthesis of selective PTP inhibitors, we now present our efforts in expansion of this concept and provide here a number of new chemical scaffolds for the development of inhibitors of different members of the PTP family. Although the core structure of these inhibitors is charged, good oral bioavailability has been observed in rat for some compounds. Furthermore, we have observed enhancement of 2-deoxy-glucose accumulation in C2C12 cells with prodrug analogues.

Structure determination of T cell protein-tyrosine phosphatase.

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.