Structure-Guided Elaboration of a Fragment-Like Hit into an Orally Efficacious Leukotriene A4 Hydrolase Inhibitor.

Leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have provided strong evidence that LTA4H is an attractive drug target for the treatment of chronic inflammatory diseases. Here, we describe the transformation of compound 2, a fragment-like hit, into the potent inhibitor of LTA4H 3. Our strategy involved two key steps. First, we aimed to increase the polarity of fragment 2 to improve its drug-likeness, particularly its solubility, while preserving both its promising potency and low molecular weight. Second, we utilized structural information and incorporated a basic amino function, which allowed for the formation of an essential hydrogen bond with Q136 of LTA4H and consequently enhanced the potency. Compound 3 exhibited exceptional selectivity and showed oral efficacy in a KRN passive serum-induced arthritis model in mice. The anticipated human dose to achieve 90% target engagement at the trough concentration was determined to be 40 mg administered once daily.

LTA4H inhibitor LYS006: Clinical PK/PD and safety in a randomized phase I clinical trial.

LYS006 is a novel, highly potent and selective, new-generation leukotriene A4 hydrolase (LTA4H) inhibitor in clinical development for the treatment of neutrophil-driven inflammatory diseases. We describe the complex pharmacokinetic to pharmacodynamic (PD) relationship in blood, plasma, and skin of LYS006-treated nonclinical species and healthy human participants. In a randomized first in human study, participants were exposed to single ascending doses up to 100 mg and multiple ascending doses up to 80 mg b.i.d.. LYS006 showed rapid absorption, overall dose proportional plasma exposure and nonlinear blood to plasma distribution caused by saturable target binding. The compound efficiently inhibited LTB4 production in human blood and skin blister cells, leading to greater than 90% predose target inhibition from day 1 after treatment initiation at doses of 20 mg b.i.d. and above. Slow re-distribution from target expressing cells resulted in a long terminal half-life and a long-lasting PD effect in ex vivo stimulated blood and skin cells despite low plasma exposures. LYS006 was well-tolerated and demonstrated a favorable safety profile up to highest doses tested, without any dose-limiting toxicity. This supported further clinical development in phase II studies in predominantly neutrophil-driven inflammatory conditions, such as hidradenitis suppurativa, inflammatory acne, and ulcerative colitis.

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Absorption, Distribution, Metabolism, and Excretion of [14C]iptacopan in Healthy Male Volunteers and in In Vivo and In Vitro Studies.

Iptacopan (LNP023) is an oral, small-molecule, first-in-class, highly potent proximal complement inhibitor that specifically binds factor B and inhibits the alternative complement pathway. Iptacopan is currently in development as a targeted treatment of paroxysmal nocturnal hemoglobinuria and multiple other complement-mediated diseases. In this study, the absorption, distribution, metabolism, and excretion (ADME) of iptacopan was characterized in six healthy volunteers after a single 100 mg oral dose of [14C]iptacopan. This was supplemented with an in vivo rat ADME study and metabolite exposure comparisons between human, rat, and dog, in addition to in vitro assays, to better understand the clearance pathways and enzymes involved in the metabolism of iptacopan. The fraction of [14C]iptacopan absorbed was estimated to be about 71%, with a time to maximum concentration of 1.5 hours and elimination half-life from plasma of 12.3 hours. Following a single dose of [14C]iptacopan, 71.5% of the radioactivity was recovered in feces and 24.8% in urine. [14C]iptacopan was primarily eliminated by hepatic metabolism. The main biotransformation pathways were oxidative metabolism via CYP2C8, with M2 being the major oxidative metabolite, and acyl glucuronidation via UGT1A1. The two acyl glucuronide metabolites in human plasma, M8 and M9, each accounted for ≤ 10% of the total circulating drug-related material; systemic exposure was also observed in toxicology studies in rat and dog, suggesting a low risk associated with these metabolites. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan. SIGNIFICANCE STATEMENT: We characterized the pharmacokinetics, excretion, metabolism and elimination of [14C]iptacopan (an oral, selective small-molecule inhibitor of factor B) in healthy human subjects. [14C]iptacopan was primarily eliminated by metabolism. The primary biotransformation pathways were oxidative metabolism via CYP2C8 and acyl glucuronidation via UGT1A1. Direct secretion of iptacopan into urine and potentially bile represented additional elimination mechanisms. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan.

Silencing of proinflammatory NF-κB and inhibition of herpes simplex virus (HSV) replication by ultrasmall gold nanoparticles (2 nm) conjugated with small-interfering RNA.

Azide-terminated ultrasmall gold nanoparticles (2 nm gold core) were covalently functionalized with alkyne-terminated small-interfering siRNA duplexes by copper-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry). The nanoparticle core was visualized by transmission electron microscopy. The number of attached siRNA molecules per nanoparticle was determined by a combination of atomic absorption spectroscopy (AAS; for gold) and UV-Vis spectroscopy (for siRNA). Each nanoparticle carried between 6 and 10 siRNA duplex molecules which corresponds to a weight ratio of siRNA to gold of about 2.2 : 1. Different kinds of siRNA were conjugated to the nanoparticles, depending on the gene to be silenced. In general, the nanoparticles were readily taken up by cells and highly efficient in gene silencing, in contrast to free siRNA. This was demonstrated in HeLa-eGFP cells (silencing of eGFP) and in LPS-stimulated macrophages (silencing of NF-κB). Furthermore, we demonstrated that nanoparticles carrying antiviral siRNA potently inhibited the replication of Herpes simplex virus 2 (HSV-2) in vitro. This highlights the strong potential of siRNA-functionalized ultrasmall gold nanoparticles in a broad spectrum of applications, including gene silencing and treatment of viral infections, combined with a minimal dose of gold.

Uptake of Functional Ultrasmall Gold Nanoparticles in 3D Gut Cell Models.

Ultrasmall gold nanoparticles (2 nm) easily penetrate the membranes of intestinal murine epithelial cells (MODE-K) and colorectal cancer cells (CT-26). They are also taken up by 3D spheroids (400 µm) of these cell types and primary gut organoids (500 µm). In contrast, dissolved dyes are not taken up by any of these cells or 3D structures. The distribution of fluorescent ultrasmall gold nanoparticles inside cells, spheroids, and gut organoids is examined by confocal laser scanning microscopy. Nanoparticles conjugated with the cytostatic drug doxorubicin and a fluorescent dye exhibit significantly greater cytotoxicity toward CT-26 tumor spheroids than equally concentrated dissolved doxorubicin, probably because they enter the interior of a spheroid much more easily than dissolved doxorubicin. Comprehensive analyses show that the cellular uptake of ultrasmall gold nanoparticles occurs by different endocytosis pathways.

Covalent Attachment of Aggregation-Induced Emission Molecules to the Surface of Ultrasmall Gold Nanoparticles to Enhance Cell Penetration.

Three different alkyne-terminated aggregation-induced emission molecules based on a para-substituted di-thioether were attached to the surface of ultrasmall gold nanoparticles (2 nm) by copper-catalyzed azide-alkyne cycloaddition (click chemistry). They showed a strong fluorescence and were well water-dispersible, in contrast to the dissolved AIE molecules. The AIE-loaded nanoparticles were not cytotoxic and easily penetrated the membrane of HeLa cells, paving the way for an intracellular application of AIE molecules, e.g., for imaging.

Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase.

The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

pH-Controlled Hierarchical Assembly/Disassembly of Multicompartment Micelles in Water.

Multicompartment micelles (MCMs) have become attractive drug delivery systems as they allow the separate storage of two or more incompatible cargos in their core compartments (e.g., drugs and dyes for imaging). A recent hierarchical self-assembly process for hydrophobic terpolymers in organic solvents showed the ability to form very homogeneous MCM populations, yet the transfer of this process into water requires a better understanding of the formation mechanism and influence of chain mobility during assembly. Here, the synthesis of a linear poly(oligo(ethylene glycol) methacrylate)-block-poly(benzyl acrylate)-block-poly(4-vinylpyridine) (POEGMA-b-PBzA-b-P4VP) triblock terpolymer by reversible addition-fragmentation chain transfer (RAFT) polymerization is reported as well as its step-wise assembly into MCMs in water with POEGMA corona, PBzA patches, and P4VP core. Reversible assembly/disassembly of the MCMs is investigated through protonation/deprotonation of the P4VP core. Interestingly, the low glass transition temperature (Tg ) of the hydrophobic PBzA middle block causes MCMs to directly disassemble into molecularly dissolved chains instead of patchy micelles due to mechanical stress from electrosteric repulsion of the protonated P4VP corona chains. In addition, pH resistant MCMs are created by core-crosslinking and fluorescent properties are added by covalent anchoring of fluorescent dyes via straightforward click chemistry.

Midostaurin, a Novel Protein Kinase Inhibitor for the Treatment of Acute Myelogenous Leukemia: Insights from Human Absorption, Metabolism, and Excretion Studies of a BDDCS II Drug.

The absorption, metabolism, and excretion of midostaurin, a potent class III tyrosine protein kinase inhibitor for acute myelogenous leukemia, were evaluated in healthy subjects. A microemulsion formulation was chosen to optimize absorption. After a 50-mg [14C]midostaurin dose, oral absorption was high (>90%) and relatively rapid. In plasma, the major circulating components were midostaurin (22%), CGP52421 (32.7%), and CGP62221 (27.7%). Long plasma half-lives were observed for midostaurin (20.3 hours), CGP52421 (495 hours), and CGP62221 (33.4 hours). Through careful mass-balance study design, the recovery achieved was good (81.6%), despite the long radioactivity half-lives. Most of the radioactive dose was recovered in feces (77.6%) mainly as metabolites, because only 3.43% was unchanged, suggesting mainly hepatic metabolism. Renal elimination was minor (4%). Midostaurin metabolism pathways involved hydroxylation, O-demethylation, amide hydrolysis, and N-demethylation. High plasma CGP52421 and CGP62221 exposures in humans, along with relatively potent cell-based IC50 for FMS-like tyrosine kinase 3-internal tandem duplications inhibition, suggested that the antileukemic activity in AML patients may also be maintained by the metabolites. Very high plasma protein binding (>99%) required equilibrium gel filtration to identify differences between humans and animals. Because midostaurin, CGP52421, and CGP62221 are metabolized mainly by CYP3A4 and are inhibitors/inducers for CYP3A, potential drug-drug interactions with mainly CYP3A4 modulators/CYP3A substrates could be expected. Given its low aqueous solubility, high oral absorption and extensive metabolism (>90%), midostaurin is a Biopharmaceutics Classification System/Biopharmaceutics Drug Disposition Classification System (BDDCS) class II drug in human, consistent with rat BDDCS in vivo data showing high absorption (>90%) and extensive metabolism (>90%).

Brain concentrations of mGluR5 negative allosteric modulator MTEP in relation to receptor occupancy--Comparison to MPEP.

BACKGROUND: To verify relation between brain free levels, receptor occupancy in vivo and in vitro affinity at the target for mGluR5 negative allosteric modulator (NAM) MTEP. METHODS: We evaluated plasma and brain extra-cellular fluid (ECF) concentration of MTEP at behaviourally active dose (5mg/kg) using in vivo microdialysis. These values were compared it to the affinity in vitro (receptor binding and FLIPR) and to receptor occupancy in vivo. Another, related substance, MPEP was used for comparison. RESULTS: MTEP and MPEP respectively inhibited mGluR5 receptors function in vitro with an affinity of 25.4 and 12.3 nM respectively. Accordingly peak ECF (extracellular fluid) levels were 1.3 and 0.14 µM, and peak total plasma levels were 7-11 and 2.6 µM. The ED50 for in vivo receptor occupancy was for both agents in the range of 0.8-0.7 mg/kg. CONCLUSIONS: At behaviourally active dose MTEP produced complete mGluR5 receptor occupancy but over 50 times higher ECF concentrations than affinity for mGluR5 receptor in vitro. This difference is seems lower for other mGluR5 NAM compounds such as MPEP. A possibly explanation could be different distribution in body compartments of both agents leading to errors of estimation with the microdialysis technique or different pharmacological activity at the receptor.

Scaffold hopping approach towards various AFQ-056 analogs as potent metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate receptor subtype 5 has evolved into a promising target for the treatment of various diseases of the central nervous system, such as Fragile X and L-DOPA induced dyskinesia. One of the most advanced clinical compound is Novartis' AFQ-056 (Mavoglurant), which served us as a template for a scaffold hopping approach, generating a structurally diverse set of potent analogs. Both the limited aqueous solubility and the relatively poor metabolic stability of AFQ-056 were improved with hexahydrocyclopenta[c]pyrrole derivative 54a, which proved to be a valuable candidate for further development.

Comparison of the mGlu(5) receptor positive allosteric modulator ADX47273 and the mGlu(2/3) receptor agonist LY354740 in tests for antipsychotic-like activity.

Recently, it has been proposed that activation of either metabotropic glutamate receptors e.g. mGlu(5) by positive allosteric modulators or stimulation of mGluR(2/3) receptors by agonists may offer new strategy in schizophrenia treatment. The aim of the present study was to compare the effect of mGlu(5) receptor positive allosteric modulator, ADX47273 (S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone), mGluR(2/3) agonist, LY354740 ((1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate) and selected neuroleptics in animal models for positive schizophrenia symptoms. ADX47273 (3 and 10mg/kgi.p.), the typical antipsychotic haloperidol (0.1 and 0.2mg/kgi.p.), the atypical antipsychotics aripiprazole (1.25-5mg/kgi.p.) and olanzapine (2.5 and 5mg/kgi.p.) all reduced amphetamine-induced hyperlocomotion in Sprague-Dawley rats, unlike the mGlu(2/3) receptor agonist LY354740 (1-10mg/kgi.p.). Interestingly, haloperidol (0.1 and 0.2mg/kgi.p.), aripiprazole (1.25-5mg/kgi.p.) and olanzapine (1.25-5mg/kgi.p.), but not ADX47273 (1-10mg/kgi.p.), all reduced spontaneous locomotion and rearings at doses effective against amphetamine-induced hyperlocomotion. This indicates that the effect of ADX47273 in combination with amphetamine may be specific, and also suggests a lack of sedative side effects. Moreover, ADX47273 (30mg/kgi.p.), haloperidol (0.1 and 0.2mg/kgi.p.) and aripiprazole (5 and 10mg/kgi.p.) reversed apomorphine (0.5mg/kgs.c.)-induced deficits of prepulse inhibition, whereas neither LY354740 (1-10mg/kgi.p.) nor olanzapine (1.25-5mg/kgi.p.) produced this effect. Lack of effect of olanzapine was unexpected and at present no convincing explanation can be provided. In conclusion, in selected rodent models for positive schizophrenia symptoms, ADX47273 showed better efficacy than LY354740.

Assessment of drug-drug interaction for silymarin.

Silymarin was assessed for drug-drug interaction by permeability studies with Caco-2 cells, for cytochrome P450 induction with human primary hepatocytes and for cytochrome P450 inhibition with human liver microsomes. Studies with Caco-2 cells revealed no interference of silymarin with the permeability of nifedipine. Silymarin did not induce cytochromes P450 2C9 and 3A4 at concentrations of 0.1; 1; and 100 microM, measured as silibinin. The inhibitory effect was tested on the nine major cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 at concentrations of 1 and 100 microM silymarin. At 1 microM concentration no or negligible inhibition of cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, and 2E1, minor inhibition of 3A4 (<20%), and moderate inhibition of 2C19 and 2D6 (<40%) were observed. Inhibition constant Ki of silymarin was determined for cytochromes P450 3A4 with 12 microM, 2C19 with 2 microM, and 2D6 with 12 microM. Only at the high concentration of 100 microM silymarin, inhibition at >50% of the cytochromes P450 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 was observed, and no or moderate inhibition was for the cytochromes P450 1A2, 2A6, and 2E1. However, in view of the clinically relevant plasma concentration of approx. 0.2 microM measured as silibinin, it is evident that there is no drug-drug interaction problem with silymarin.

Characterization of the species-specificity of peroxisome proliferators in rat and human hepatocytes.

Peroxisome proliferation is a well-defined pleiotropic effect that is mediated by the ligand inducible transcription factor peroxisome proliferator-activated receptor (PPAR) alpha. Because marked peroxisome proliferation occurs in rodents but not in humans, we aimed to elucidate the molecular and cellular determinants of this species-specificity in hepatocytes. Analysis of peroxisomal marker enzyme activities confirmed that peroxisome proliferators induced acyl-CoA oxidase (ACOX) and to a lesser extent catalase in rat hepatocytes, but not in human hepatoma HepG2 cells. Transient transfection assays revealed that ciprofibrate and Wy 14,643 induced rat but not human PPARalpha-mediated reporter gene activity in rat FAO and primary hepatocytes on rat but not on human PPARalpha response elements (PPREs). In contrast, in human HepG2 and primary human hepatocytes, peroxisome proliferators did not induce either human or rat PPARalpha activity regardless of rat or human PPRE sequences. In addition, no induction of ACOX gene expression was observed in human hepatocytes independent of the expression level of human PPARalpha. Remarkably, no distinct peroxisome proliferation related responses were observed in human hepatocytes when rat PPARalpha was transfected, although human hepatocytes were responsive to PPARalpha-mediated induction of carnitine palmitoyl transferase-1A and 3-hydroxy-3-methylglutaryl-CoA synthase. These results confirmed that PPARalpha and PPREs are important determinants for the species-specificity of peroxisome proliferation. Nevertheless, our results showed that human hepatocytes limit the extent of peroxisome proliferation regardless of PPARalpha expression.