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The role of AXIN2+ pericentral hepatocytes during liver homeostasis and regeneration
No full textThe existence of a specialized tissue stem cell niche, which safeguards liver homeostasis and repair, like AXIN2+ pericentral hepatocytes, is controversial. Using an improved AXIN2 lineage tracing model we confirm the regenerative capacity of intestinal stem cells (ISCs) but contest that pericentral hepatocytes repopulate the liver parenchyma during homeostasis. Likewise, liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver rather than by the pericentral niche. Periportal hepatocyte injury triggers local repair and auxiliary proliferation throughout all liver zones. DTA-mediated ablation of the AXIN2+ pericentral hepatocytes transiently disrupts this niche which is reestablished by conversion of glutamine synthetase (GS)- hepatocytes, newly juxtaposed to the pericentral vein, to GS+ hepatocytes rather than by proliferation of residual AXIN2+ hepatocytes. Hepatocytes throughout the liver upregulate AXIN2 and LGR5 following injury and contribute to liver regeneration on demand, without zonal dominance of a putative pericentral stem cell niche
Craig plot 2.0 - An interactive navigation in the substituent bioisosteric space
No full textBioisosteric replacement is a standard technique that is used in medicinal chemistry to design analogs of bioactive molecules with similar biological activity and with additional improved characteristics. Successful application of this technique relies on a good knowledge of properties of common organic substituents and an efficient way to navigate their space. In this study the properties of the most common substituents present in bioactive molecules are analysed and a freely-available web tool https://bit.ly/craigplot that allows visualization, analysis and selection of bioisosteric substituents is presented
A first-in-human phase 1 dose escalation study of spartalizumab (PDR001), an anti-PD-1 antibody, in patients with advanced solid tumors
No full textBackground Spartalizumab is a humanized IgG4κ monoclonal antibody that binds programmed death-1 (PD-1) and blocks its interaction with PD-L1 and PD-L2. This phase 1/2 study was designed to assess the safety, pharmacokinetics, and preliminary efficacy of spartalizumab in patients with advanced or metastatic solid tumors. Methods In the phase 1 part of the study, 58 patients received spartalizumab, intravenously, at doses of 1, 3, or 10 mg/kg, administered every 2 weeks (Q2W), or 3 or 5 mg/kg every 4 weeks (Q4W). Results Patients had a wide range of tumor types, most commonly sarcoma (28%) and metastatic renal cell carcinoma (10%); other tumor types were reported in ≤3 patients each. Most patients (93%) had received prior antineoplastic therapy (median three prior lines) and two-thirds of the population had tumor biopsies negative for PD-L1 expression at baseline. The maximum tolerated dose was not reached. The recommended phase 2 doses were selected as 400 mg Q4W or 300 mg Q3W. No dose-limiting toxicities were observed, and adverse events included those typical of other PD-1 antibodies. The most common treatment-related adverse events of any grade were fatigue (22%), diarrhea (17%), pruritus (14%), hypothyroidism (10%), and nausea (10%). Partial responses occurred in two patients (response rate 3.4%); one with atypical carcinoid tumor of the lung and one with anal cancer. Paired tumor biopsies from patients taken at baseline and on treatment suggested an on-treatment increase in CD8+ lymphocyte infiltration in patients with clinical benefit. Conclusions Spartalizumab was well tolerated at all doses tested in patients with previously treated advanced solid tumors. On-treatment immune activation was seen in tumor biopsies; however, limited clinical activity was reported in this heavily pretreated, heterogeneous population. The phase 2 part of this study is ongoing in select tumor types. Trial registration number NCT02404441
Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability
No full textChimeric antigen receptor–T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response
Nidufexor (LMB763), a Novel FXR Modulator for the Treatment of Nonalcoholic Steatohepatitis (NASH)
No full textFarnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we have described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we describe the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo. Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy
From powder to tablets: Investigation of Residence Time Distributions in a Continuous Manufacturing Process Line as Basis for Continuous Process Verification
No full textIn Continuous Manufacturing (CM), process units are directly connected to each other forming a continuous process line. In combination with suitable on-line process analytical technologies, CM enables the dynamic manipulation of process parameters in response to the current observed process conditions and hence, facilitates real-time process control. However, the implementation of such a control strategy is only feasible, if the overlying control system knows precisely what material is when and where at all times. Consequently, thorough knowledge of the residence time distribution (RTD) of the material throughout the whole manufacturing process needs to be established early on in development. Once RTD is well understood, its constant observation could also be used for continuous process verification purposes. As continuous processes that run over extended periods of time are susceptible to unforeseen incidents like equipment wear-out or clogging, drifts or shifts in RTD could indicate such issues early on.
The presented work aims to demonstrate this proposed concept for an integrated wet-granulation CM process. To achieve this aim, three steps were completed: First, thorough RTD knowledge was generated, by inducing step-tests in active pharmaceutical ingredient (API) content in the range of ±30% at varying process conditions, and analyzing the material RTDs via NIRS analysis at four different locations in the line. Second, it was demonstrated that also low-level step tests of ±5% and even ±3% are sufficient for accurate RTD determination. This validated the possibility of continuous RTD assessment during (pre-)validation trials or even commercial manufacturing, as the drug product would comply with required quality characteristics (content uniformity, Assay). In the third step it was then demonstrated that recurring low-level step testing during routine manufacturing could be used as a way to determine the current system health, as observed changes in RTD indicated blockages and material hold-up in the line. And while deliberate changes in API content during commercial production might seem counter intuitive, they would actually aid in ensuring the production of quality product in a better way, than running at constant process settings over an extended period of time
Synthesis of 5-iodo-7-aza-indazole by Diels–Alder cascade and se-lected examples of C–C and C–N cross-coupling reactions
No full textAlthough pyrimidines are not among the most reactive partners in intramolecular inverse-electron demand [4s+2s] reactions with alkynes, they could be activat-ed under mild and practical conditions, leading to fused nitrogen-containing heterocycles. We report an opti-mized synthesis of a 5-iodo-7-aza-indazole by a one-pot Diels–Alder cascade that starts from a pyrimidine sub-stituted in the 2-position by an (alkynyl)hydrazone. The safety of the process and the environmental impact were thoroughly evaluated. Eventually, a selection of cross-coupling reactions of 17 was studied and allowed the introduction of carbon- and nitrogen-based nucleophiles at the C5-position in good to excellent yields
A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL
No full textTargeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner. However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins
Stabilizing inactive conformations of the paracaspase MALT1 as an effective approach to inhibit its protease activity
No full textThe paracaspase MALT1 (mucosa associated lymphoid tissue lymphoma translocation protein 1) plays an important role in various immune pathways and has been proposed as a therapeutic target for auto-immune disorders as well as cancers (i.e. DLBCL). We explored different mechanisms to inhibit the protease activity of MALT1 and discovered two unrelated chemical scaffolds. Biophysical and structural studies revealed that both scaffolds stabilize the protease in an inactive conformation. While one ligand binds to the allosteric site at the interface between the caspase and the Ig3 domain, the other ligand binds to the active site in a so far undescribed mechanism. Iterative structure based drug discovery on one scaffold resulted in the identification of a potent, selective and bioavailable MALT1 inhibitor
Absorption, Distribution, Metabolism, and Excretion (ADME) of Capmatinib (INC280) in Healthy Male Volunteers and In Vitro Aldehyde Oxidase Phenotyping of the Major Metabolite
No full textCapmatinib (INC280), a highly selective and potent inhibitor of the MET receptor tyrosine kinase, has demonstrated clinically meaningful efficacy and a manageable safety profile in patients with advanced NSCLC harboring MET exon 14 skipping mutations. We investigated the absorption, distribution, metabolism, and excretion of capmatinib in six healthy male volunteers after a single peroral dose of 600 mg 14C-labeled capmatinib. The mass balance, blood and plasma radioactivity, and plasma capmatinib concentrations were determined along with metabolite profiles in plasma, urine, and feces. The metabolite structures were elucidated using mass spectrometry and comparing with reference compounds. The parent compound accounted for most of the radioactivity in plasma (42.9 ± 2.9%). The extent of oral absorption was estimated to be 49.6%; the maximum concentration (Cmax) of capmatinib in plasma was reached at 2 h (median Tmax). The apparent mean elimination half-life of capmatinib in plasma was 7.84 h. Apparent distribution volume (Vz/F) of capmatinib during the terminal phase was moderate to high (geometric mean 473 L). Metabolic reactions involved lactam formation, hydroxylation, N-dealkylation, formation of a carboxylic acid, hydrogenation, N oxygenation, glucuronidation, and combinations thereof. The most abundant metabolite, M16 was formed by imidazo-triazinone formation (lactam formation). Absorbed capmatinib was eliminated mainly by metabolism and subsequent biliary/fecal and renal excretion. Excretion of radioactivity was complete after 7 days. In vitro studies demonstrated that CYP3A was the major P450 enzyme subfamily involved in hepatic microsomal metabolism, and M16 formation was mainly catalyzed by cytosol aldehyde oxidase