SXN101959, a targeted secretion inhibitor, has the same relative potency compared to GHRH(1-44) at GHRH receptors (GHRH-R) of rat, human and macaque

Mark Elliott, Elena Fonfria, Stephen Johnstone, Fraser Hornby, Sarah Donald, Elaine Harper. Syntaxin Ltd, units 4-10, the quadrant, abingdon, oxford, UK.

Syntaxin Ltd has developed a new class of targeted secretion inhibitor (TSI) by the generation of novel multi-domain recombinant proteins rationally engineered from Clostridium botulinum toxin in which the normal neuronal targeting is changed to direct the TSI to specific cell types with aberrant secretion (Foster & Chaddock, 2010). The TSI SXN101959 has been designed to specifically bring about inhibition of growth hormone (GH) secretion from pituitary somatotrophs for the treatment of acromegaly; a disease caused by excess secretion of GH from the pituitary. TSIs bring about an inhibition of secretion through a combined activity of their three domains. In the case of SXN101959, the targeting domain binds to and activates GHRH-R to effect internalization of SXN101959 into endosomes of the somatotroph. The translocation domain facilitates transport of the third and final domain across the endosomal membrane into the cytosol. The third domain is a metalloprotease that cleaves specific SNARE (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein) proteins which are critical to the process of vesicular secretion. In this study we characterise the pharmacology of SXN101959 relative to GHRH(1-44) at recombinant GHRH-R of rat and macaque in order to establish whether these are relevant species for establishing the in-vivo efficacy and safety of this TSI. The pharmacology at the human orthologue has also been examined.

The pharmacology of SXN101959 at GHRH-R of each species was assessed relative to GHRH(1-44) using accumulation of cAMP as an indicator of receptor activation. CHO-K1 or GH3 cells expressing recombinant receptors were incubated at 21 °C (rat, GH3-rGHRH-R, 120min; human, CHO-K1-hGHRH-R, 90min; macaque, CHO-K1-mGHRH-R, 150min) with increasing concentrations of SXN101959 or GHRH(1-44). cAMP was measured using a TR-FRET immunoassay. The effect of both ligands on cAMP accumulation in wild type (WT) CHO-K1 and GH3 cells was also determined. That the SXN101959-induced cAMP accumulation was GHRH-R mediated was confirmed through pre-incubation of cells (30min) with a selective GHRH-R antagonist (JV-1-36, Varga et al., 1999; pKB = 8.7) before incubation with increasing concentrations of ligand.

In WT CHO-K1 and GH3 cells, GHRH(1-44) and SXN101959 had no effect on cAMP accumulation at concentrations up to 0.3 and 1µM, respectively. In contrast, both GHRH(1-44) and SXN101959 produced a concentration-dependent increase in cAMP accumulation in cells expressing GHRH-R. The potency (pEC50, mean ± s.e.mean) of SXN101959 was 7.90 ± 0.06 (n = 5), 7.52 ± 0.04 (n = 5) and 7.37 ± 0.03 (n = 4) at rat, human and macaque GHRH-R, respectively. There was no significant difference in the relative potency of SXN101959 compared to GHRH(1-44) at the three receptor orthologues (pEC50 GHRH(1-44)/ pEC50 SXN101959; rat = 1.14 ± 0.01, human = 1.12 ± 0.01, macaque = 1.16 ± 0.01; p>0.05, one-way ANOVA). In addition, the Emax of SXN101959 at the three receptor orthologues was not significantly less than that produced by GHRH(1-44) (paired t-test, p>0.05). The GHRH-R antagonist JV-1-36 produced a significant rightward-shift (mean shift log units ± s.e.mean) in the concentration-effect curve of SXN101959 at the rat (0.68 ± 0.24; n = 5), human (0.69 ± 0.21; n = 5) and macaque (0.64 ± 0.04; n = 6) GHRH-R (paired t-test, p<0.05).

These studies demonstrate that SXN101959 is an equipotent full agonist at GHRH-R of rat, human and macaque and that the rat and macaque are relevant species for gaining an understanding of the in-vivo efficacy and safety of this TSI.