IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.

Nanobodies are antigen-binding, single variable domain proteins derived from naturally-occurring, heavy chain only antibodies. They are highly soluble, stable, and can be linked to build multi-specific formats. Several Nanobodies are currently in clinical development in different therapeutic areas, for both chronic and acute applications. For the former, prolonged exposure is achieved by half-life extending moieties that target endogenous albumin, while for the latter, non-half-life extended constructs are preferable. To demonstrate the general pharmacokinetic behavior of both formats, serum levels of seven intravenously administered Nanobodies were analyzed in cynomolgus monkeys, mice or rabbits. In monkeys, the total clearance of a monomeric irrelevant Nanobody was rapid (2.0 mL/(min*kg)) and approximated the species glomerular filtration rate, indirectly suggesting that the Nanobody was mainly eliminated via the kidneys. When linked to an anti-albumin Nanobody, a 376-fold decrease in clearance was observed, resulting in a terminal half-life of 4.9 days, corresponding to the expected species albumin half-life. Similar conclusions were drawn for (non-) half-life extended mono-, bi-and trimeric Nanobodies in mice or rabbits, suggesting that these kinetic principles apply across species. Applying this knowledge to species translation and study design is crucial for successful pre-clinical development of novel therapeutic Nanobody candidates.

Regorafenib is an oral multikinase inhibitor with clinical efficacy in a range of advanced solid tumours. A population pharmacokinetic (PK) model was developed to evaluate the variability of the PK of regorafenib and its pharmacologically active metabolites M-2 and M-5 in solid tumours. Methods: The model was initially developed using densely sampled phase 1 data and information on food intake to incorporate enterohepatic circulation (EHC) that was identified to considerably contribute to the PK of regorafenib. This was then applied to sparsely sampled data from four phase 3 studies in patients with advanced solid tumours. The need for exact food intake data to estimate individual drug exposure was evaluated. Results: By incorporating EHC, the model adequately described the PK profiles of regorafenib, M-2 and M-5 after single and multiple doses in patients from phase 1 studies. Individual exposure in phase 3 studies was adequately described based on assumptions on the time and frequency of food intake, although exact food intake data are recommended to improve the estimation. Covariate analysis identified sex and body mass index (BMI) as impacting exposure to regorafenib, and sex as strongly impacting exposure to M-2 and M-5 (also influenced by the BMI effect on parent regorafenib in the joint model developed); however, these factors accounted for a small portion of the overall variability in exposure. Conclusions: The adequate description of regorafenib PK after multiple dosing requires the incorporation of EHC. Neither single nor combined covariates predicted exposures that would warrant a priori regorafenib dose adjustment.

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ASP8232 is a novel inhibitor of vascular adhesion protein-1 that was under evaluation for reducing residual albuminuria in patients with diabetic kidney disease. To characterize the pharmacokinetics (PK) of ASP8232 and its effect on vascular adhesion protein 1 (VAP-1) plasma activity and VAP-1 concentrations (pharmacodynamics, PD) in an integrated and quantitative manner, a target mediated drug disposition model was developed based on pooled data from four completed clinical trials with ASP8232 in healthy volunteers, and in patients with diabetic kidney disease and diabetic macular edema, respectively. In this model, the binding of ASP8232 to its soluble and membrane-bound target in the central and peripheral compartments were included. The model was able to adequately describe the non-linear PK and PD of ASP8232. The observed difference in PK between healthy volunteers and renally impaired patients could be explained by an effect of baseline estimated glomerular filtration rate on ASP8232 clearance and relative bioavailability. The relationship between ASP8232 concentration and VAP-1 inhibition was successfully established and can be applied to simulate drug exposure and degree of VAP-1 inhibition for any given dose of ASP8232 across the spectrum of renal function.