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Validation of the salivary tracer technique for the determination of the in vivo disintegration of hard gelatin capsules
No full textThe process of disintegration is a crucial step in oral drug delivery with immediate release dosage forms. In this work, the salivary tracer technique was applied as a simple and inexpensive method for the investigation of the in vivo disintegration time of hard gelatin capsules filled with caffeine. The disintegration times observed with the salivary tracer technique were verified by magnetic resonance imaging (MRI). After an overnight fast of at least 10 h and caffeine abstinence of minimum 72 h, conventional hard gelatin capsules containing 50 mg caffeine and 5 mg iron oxide were administered to 8 healthy volunteers. For the period of 1 h after capsule intake, subjects were placed in supine position in the MRI scanner, and scans were performed in short time intervals. Each MRI measurement was directly followed by saliva sampling by drooling. Salivary caffeine concentrations were determined by high performance liquid chromatography followed by mass spectrometric detection (LC/MS-MS). The time point of capsule disintegration was determined by visual inspection of the MR images as well as by an increase in the salivary caffeine concentration. The results indicated that the difference in mean disintegration times of the capsules as determined by the two in vivo methods was around 4 min (8.8 min for MRI vs 12.5 min for saliva). All disintegration times determined by the salivary tracer technique were slightly higher. This delay could be explained by the fact that the appearance of caffeine in saliva required drug absorption in the small intestine. Because capsule disintegration happened mainly in the stomach, the exact site of disintegration as well as the processes of gastric mixing and gastric emptying contributed to the delay between the two methods. This work demonstrated the feasibility of the salivary tracer technique to investigate the in vivo disintegration of immediate release dosage forms in a simple and reliable manner
A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2
No full textDespite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy
Design, synthesis and pre-clinical characterization of selective Factor D inhibitors targeting the alternative complement pathway
No full textComplement Factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (MPGNII) and paroxysmal nocturnal hemoglobinuria (PNH). Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide (LPS)-induced AP activation model, as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD
Pharmacological profiling of a dual FAK/IGF-1R kinase inhibitor TAE226 in cellular and in vivo models
No full textObjective: A dual inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor 1 receptor (IGF-1R), TAE226, was evaluated in a panel of cancer cell lines, MIA PaCa-2 human pancreatic tumor and 4T1 murine breast tumor models. The profiling data were generated during the drug discovery research prior to the first publication of TAE226 appeared in 2007 [1-3].
Results: In a panel of 37 cancer cell lines, TAE226 showed a mean GI50 value of 0.76 mole/L and was not a substrate of P-glycoprotein. In the MIA PaCa-2 model, TAE226 inhibited phosphorylation of FAK at Y397 and as IGF-1R signaling, phosphorylation of Akt at S473 in the cell culture in vitro and the tumor in vivo. Oral administration of TAE226 induced tumor stasis at 30 mg/kg and tumor regression at 100 mg/kg in the subcutaneous tumor, and inhibited the orthotopic tumor growth in a dose-dependent manner. Similarly in the 4T1 model, TAE226 inhibited phosphorylation of FAK at Y397 and Akt at S473 in the cell culture in vitro and the tumor in vivo. Oral administration of TAE226 inhibited the orthotopic tumor growth and metastasis to the lung in a dose-dependent manner. Thus, the dual FAK/IGF-1R kinase inhibitor TAE226 would be a potential therapeutic agent to prevent tumor metastasis as well as progression
Rethinking drug design in the artificial intelligence era
No full textArtificial intelligence (AI) tools are increasingly being applied in drug discovery. While some protagonists point to vast opportunities potentially offered by such tools, others remain sceptical, waiting for a clear impact to be shown in drug discovery projects. The reality is probably somewhere in-between these extremes, yet it is clear that AI is providing new challenges not only for the scientists involved but also for the biopharma industry and its established processes for discovering and developing new medicines. This article presents the views of a diverse group of international experts on the ‘grand challenges’ in small-molecule drug discovery with AI and the approaches to address them
Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data
No full textThe bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line ″clonal origin″ an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal-derivation 1-4. However, older production cell lines developed before the implementation of these methods, herein referred to as ″legacy cell lines″, may not meet current regulatory expectations for demonstration of clonal-derivation. In this article, the members of the IQ Consortium ″Working Group on Clonality″ present our position that the demonstration of process consistency and product comparability of critical quality attributes (CQAs) throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal-derivation standards. With this commentary we discuss advantages and limitations of genetic testing methods to support clonal-derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to IND application and prior to demonstration of product and process consistency at BLA submission
Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of an Added-Value Chemical
No full textNon-heme iron halogenases represent synthetically valuable biocatalysts that are capable of halogenating unactivated sp3-hybridized carbon centers with exquisite stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. Here we engineered the recently discovered halogenase WelO5* for chlorination of a pharmaceutically interesting martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wild-type enzyme was generated. Moreover, we identified key positions in the active site which allowed directing the halogen to different positions in our target substrate. This report provides the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for the late-stage halogenation of value-added chemicals
Design and Discovery of N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide (LXH254), A selective, efficacious, well-tolerated RAF inhibitor targeting RAS mutant cancers: The path to the clinic
No full textRAS and BRAF oncogenes are mutated in more than one-third of human cancers and exquisite dependency on CRAF, MEK1/2 and ERK1/2 has been demonstrated in preclinical models of RAS mutant cancer. Direct pharmacological inhibition of RAS has remained elusive and efforts to target CRAF have been challenging due to the nature of the RAF signaling complex downstream of activated RAS and the poor overall kinase selectivity profile of putative RAF inhibitors such as sorafenib and RAF265. Herein, we describe 15 (LXH254), a selective B/C RAF inhibitor, which has been developed through a hypothesis-driven approach focusing on drug-like properties. We have previously disclosed the discovery of 3 (RAF709), a preclinical tool compound which was potent, selective, efficacious, and well-tolerated in preclinical models, but the high intrinsic clearance [HLM Cl(int) = 94] precluded further development.X The high clearance of 3 by HLM prompted the medicinal chemistry team to further investigate close analogs as well as novel scaffolds. While keeping drug-like properties in mind, the team identified multiple cell-potent scaffolds with low-to-moderate human clearance and progressed them into in-vivo pharmacology studies. Unexpectedly, the majority of novel scaffolds caused significant body weight loss in mice for unknown reasons, with the 2-pyridine series emerging as the only scaffold which was not plagued by this problem. A structure-based approach led to the realization that an alcohol side-chain in the 2-position of the pyridine could interact with the DFG loop and significantly improve cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition of CYP3A4 (TDI) led to the discovery of 15, which had favorable PK and proved to be efficacious in multiple xenograft models such as Calu-6 (KRASQ61K),with a favorable therapeutic index. Due to its excellent in vitro/ in vivo properties, it has progressed through preclinical toxicology studies and now being tested as a single agent and as a combination partner in phase 1 clinical trial
Biosynthesis of fragin is controlled by a novel quorum sensing signal
No full textMembers of the diazenium diolate class of natural compounds show great
potential for drug development. Yet, their biosynthesis has remained elusive to
date. Here we identify a gene cluster directing the biosynthesis of the diazenium
diolate compound fragin in the opportunistic pathogen Burkholderia
cenocepacia H111. Fragin is shown to be the major antifungal compound
produced by this bacterium. We also show that a subset of the fragin
biosynthetic genes is involved in the synthesis of a novel cell-to-cell signaling
molecule, valdiazen. RNA-Seq analysis revealed that valdiazen not only controls
its own and fragin biosynthesis but is a global regulator of more than 150 genes.
Homologs of the valdiazen biosynthesis genes are found in various bacteria
suggesting that valdiazen is the first member of a new class of signal molecules.
Using structural information, in silico prediction of enzymatic functions and
biochemical data we propose a biosynthesis route for fragin and valdiazen
The landscape of cancer cell line metabolism
No full textDespite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography–mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism