BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis.

BACKGROUND: Airway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583. METHODS: Binding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model. RESULTS: BI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma. CONCLUSION: BI 1291583-mediated inhibition of CatC is expected to restore the protease-antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Quantitative Expression of Hepatobiliary Transporters and Functional Uptake of Substrates in Hepatic Two-Dimensional Sandwich Cultures: A Comparative Evaluation of Upcyte and Primary Human Hepatocytes.

Deficient functional expression of drug transporters incapacitates most hepatic cell lines as a reliable tool for evaluating transporter-mediated drug-drug interactions. Recently, genetically modified cells (referred to as upcyte hepatocytes) have emerged as an expandable, noncancerous source of human hepatic cells. Herein, we quantified mRNA and protein levels of key hepatobiliary transporters and we assessed associated uptake activity in short- and long-term cultures of upcyte human hepatocytes (UHH) in comparison to cryopreserved primary human hepatocytes (cPHH). Expression of canalicular efflux pumps, such as MRD1/ABCB1, MATE1/SLC47A1, and MRP2/ABCC2, was relatively well preserved in UHH. By contrast, long-term cultivation of UHH in a two-dimensional sandwich configuration [sandwich-cultured upcyte human hepatocytes (SCUHH)] was required to upregulate organic anion-transporting polypeptide OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, NTCP/SLC10A1, and OCT1/SLC22A1 mRNA expression, which correlated well with respective protein abundances. However, mRNA and protein levels of sinusoidal solute carrier transporters, except for NTCP and OATP2B1, remained low in SCUHH compared to sandwich-cultured cPHH. OCT1- and NTCP-mediated uptake of N-methyl-4-phenylpyridinium acetate and taurocholate was demonstrated in both hepatic models, whereas active uptake of OATP1B1/1B3-selective marker substrates, paralleled by markedly reduced SLCO1B1/1B3 expression, were not detectable in SCUHH. Uptake studies under Na+-depletion and excess of taurocholate confirmed the presence of functional NTCP protein and indicated that NTCP, apart from OATP2B1, contributed substantially to the overall hepatic uptake of rosuvastatin in SCUHH. In conclusion, our data suggest that SCUHH, despite their limitation for evaluating OATP1B1/1B3-mediated transport processes, retain NTCP, OATP2B1, and OCT1 transport activities and thus may be considered as a tool for elucidating compensatory uptake pathways for OATP1B1/1B3 substrates.

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds.

In vitro models based on primary human hepatocytes (PHH) have been advanced for clearance (CL) prediction of metabolically stable compounds, representing state-of-the-art assay systems for drug discovery and development. Yet, limited cell availability and large interindividual variability of metabolic profiles remain shortcomings of PHH. Upcyte human hepatocytes (UHH) represent a novel hepatic cell system derived from PHH, exhibiting proliferative capacity for approximately 35 population doublings. UHH from three donors were evaluated during culture for up to 18 days, investigating relative mRNA expression and in situ enzyme activity of cytochrome P450s (P450s), UDP-glucuronosyltransferases, and sulfotransferases. Furthermore, UHH were used for predicting hepatic CL of 21 marketed low to intermediate CL drugs. In a typical experiment, expansion from 3.9 × 10(6) up to 8.5 × 10(7) cells was achieved during subculture. When maintained at confluence, transcripts of major P450s were expressed at donor-specific levels with sustained activities for the majority of isoforms, showing generally low CYP1A2 and high CYP2B6 activity levels. For donor 151-03, CL prediction based on depletion experiments resulted in an average fold error of 2.0, and 80% of compounds being predicted within twofold to in vivo CL for a subset of 10 low CL drugs. UHH showed sustained and consistent activity of drug-metabolizing enzymes (DME), resulting in highly reproducible CL prediction performance. In conclusion, UHH show promising potential as alternative to PHH for standardized in vitro applications in discovery research based on their stable, hepatocyte-like DME phenotype and virtually unlimited cell availability.

An orally bioavailable positive allosteric modulator of the mGlu4 receptor with efficacy in an animal model of motor dysfunction.

A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure-activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.