The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovirresistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen in immunocompromised individuals, including transplant recipients and tumor or AIDS patients, and remains the leading viral cause of birth defects (1,9,12,17,29). To date, a limited number of drugs are licensed for the systemic treatment of HCMV infection and disease: ganciclovir (GCV) (Cymevene; Roche), its oral prodrug valganciclovir (VGCV) (Valcyte; Roche), cidofovir (CDF) (Vistide; Gilead), and foscarnet (FOS) (Foscavir; Astra-Zeneca). In addition, valaciclovir (VACV) (Valtrex; GlaxoSmithKline), a drug that has been primarily developed for the treatment of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infection, has gained marketing approval in certain countries for prophylaxis of HCMV infections in transplant patients. Although GCV, VGCV, CDF, and FOS are effective, several drawbacks are associated with the use of these drugs, including toxicity, poor oral bioavailability (except VGCV), and emergence of drug resistance (3,20). The active forms of GCV, CDF, and FOS share the same molecular target, the viral polymerase UL54. Consequently, drug-resistant strains of HCMV encoding UL54 mutations have been found for all three compounds, and the emergence of cross-resistant strains has been described in clinical settings. In addition, resistance to GCV is also associate...

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent IntroductionThe successive adhesion, activation, and aggregation of platelets are key processes in arterial thrombus formation after endothelial damage. 1,2 Both rupture of atherosclerotic plaques as well as surgical interventions to treat atherosclerosis (eg, percutaneous coronary intervention [PCI]) may cause exposure of the subendothelium and subsequent clot formation. Eventually, this can result in the occlusion of arteries, leading to ischemia, myocardial infarcts, or stroke. Given the central role of platelets in thrombosis, a substantial number of currently marketed antithrombotic drugs, such as aspirin, clopidogrel, and abciximab, target different steps involved in platelet activation and aggregation. 1,2 Thanks to their complementary mechanisms of action, the combination of these agents inhibits platelet aggregation to a greater extent than either agent alone. 3 However, the use of these antiplatelet drugs is hampered by an increased bleeding risk 1,2 and the occurrence of treatment resistance in some patients. 4 Moreover, the irreversible nature of their action can complicate the staunching of bleeding. 1,2 Inhibition of the initial adhesion of platelets to subendothelial collagen provides an alternative strategy to prevent unwanted clot formation. The plasma glycoprotein VWF plays a pivotal role in this adhesion via binding to exposed collagen on the one hand, and the interaction of its A1 domain with the gpIb-IX-V receptor complex on the surface of platelets on the other hand. [5][6][7] Interestingly, the VWF A1 domain is only exposed under high-shear conditions, 8,9 so VWF only acts as a bridging molecule between collagen and platelets in small or stenosed arteries. Therefore, it is expected that drugs inhibiting this interaction between VWF and platelets show an improved safety profile with respect to bleeding tendency. Indeed, the antithrombotic effect of several compounds targeting the gpIb-VWF-A1-axis, like aurintricarboxylic acid, 10-12 recombinant VWF fragments, 10,13-16 a recombinant gpIb chimeric protein, 17,18 anti-VWF mAbs, [19][20][21][22][23][24][25][26][27] and an anti-VWF aptamer 28 has been demonstrated in vitro and in vivo, without increasing the bleeding risk. 13,[16][17][18]21,23,25,28,29 Nevertheless, until now only 3 drug candidates have been evaluated in humans, including ALX-0081. [30][31][32][33] We developed ALX-0081, a bivalent humanized Nanobody directed against the A1 domain of VWF. Nanobodies are therapeutic proteins derived from the heavy-chain variable domains (VHH) that occur naturally in heavy-chain-only Igs of Camelidae. 34,35 Here we...

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.