Assessment of the in vitro metabolic stability of CEP-37440, a selective FAK/ALK inhibitor, in HLMs using fast UPLC–MS/MS method: in silico metabolic lability and DEREK alerts screening
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CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism.Keywords:
Lability
Metabolic stability
In vitro metabolic stability testing of phosphorothioate 2'-O-methoxyethyl (2'-MOE) partially modified antisense oligonucleotides (ASOs) is not routinely performed to help screen discovery compounds (eg, predict in vivo half-lives), as no suitable in vitro test system currently exists. The aims of this work were to develop, optimize, and evaluate an in vitro whole liver homogenate (rat or human) test system. The test system was used to evaluate in vitro metabolic stabilities (intrinsic clearance) of selected ASOs, with results compared to reported in vivo half-lives, and generated metabolites also identified. Test system optimization involved preincubating whole liver homogenates at 37°C for ≥24 hours, which increased in vitro ASO metabolism rate. From calculated in vitro intrinsic clearance (CL(int)) values in preincubated rat or human whole liver homogenates, metabolic stabilities of fully phosphorothioated 2'-MOE ASOs (ISIS 104838 and ISIS 301012) were, as expected, greater (ie, lower CL(int)) than a 2'-MOE ASO containing a single phosphodiester substitution (ISIS 104838PO10). However, comparable-to-lower in vitro metabolic stability for ISIS 301012 was seen compared to ISIS 104838, in contrast to reported ∼2-fold longer in vivo tissue elimination half-lives for ISIS 301012. Identified in vitro metabolic products of ISIS 301012 were consistent with previously reported in vivo observations.
Metabolic stability
Phosphodiester bond
Metabolic pathway
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The strategy to screen compounds solely for pharmacological potency and selectivity in the early stages of drug discovery brought the pharmaceutical industry to face the stark reality of disproportionate attrition later in the development stage due to poor drug disposition characteristics. This attrition contributed to the exorbitant costs of discovering and developing drugs. Considering ADME (Absorption, Distribution, Metabolism, and Excretion) characteristics of compounds early in the discovery process can wisely direct resources to compounds that have greater potential to survive the clinical trial stages of drug development. However, experimental determination of ADME characteristics is not practical for large numbers of compounds. Therefore, focus is being centered on bringing in silico approaches earlier in the discovery process to assess ADME properties solely from molecular structure. Given that metabolism is one of the most important of the ADME properties, in this paper we review a number of metabolism in silico tools and models that have potential applications in drug discovery. We then describe a step-by-step process, as practiced in our laboratories, to construct and deploy reliable in silico metabolic stability and other ADME screens. Additionally, we give examples of the application of our metabolic stability in silico screens in scaffold selection, ADME space enrichment, and rationalizing synthesis and testing of compounds in the drug discovery process. Agreements between the experimental and in silico metabolic stability values ranging from 84% to 100% have convinced many discovery project teams to routinely use these in silico models. Finally, we present our ideas on the successful implementation of in silico models and tools for significant impact on drug discovery and development Keywords: In silico, ADME, metabolism, metabolic stability, drug discovery, QSAR models, ADME software
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Metabolic stability
Drug Development
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Lability
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The study of P-lability in languages in which the relationship between transitive and intransitive predication can be characterized as radical P-alignment must take into account the formal distinction between weak and strong lability, and the semantic distinction between argument structure modifying and argument structure preserving lability. Radical P-alignment is particularly common among Daghestanian languages in which some authors operating with a loose definition of P-lability have argued that P-lability is pervasive, whereas others have argued that, in the same languages, P-lability is exceptional. On the basis of more precise definitions, it is shown that, in the languages in question, all transitive verbs exhibit a behavior whose characterization as a type of lability may be controversial, depending on the definition of lability, whereas some of them only show a behavior that stands closer to prototypical lability. This paper argues that the observation of causativization is particularly relevant to the analysis of lability in such languages.
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Argument (complex analysis)
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The degree of lability of a given metal complex species is modified in the presence of a mixture of ligands. This modification is a consequence of the coupling of the association and dissociation processes of all of the complexes according to the competitive complexation reaction scheme. We show that, because of the mixture effect, the lability of a given complex usually increases when another more labile complex is added into the system, while it decreases upon addition of a less labile one. Typically, complexes tend to adapt to the global lability of the mixture. A quantitative evaluation of these effects for diffusion-limited conditions in a finite domain by rigorous numerical simulation in a system with two complexes indicates that the lability degree of a complex can change by more than 100% with respect to that in the single ligand system. The impact of the mixture effect on the metal flux depends at least on two main factors: the respective abundance of the metal species and the particular values of their lability degrees. Dominant complexes (i.e., those most abundant when these complexes have equal diffusion coefficients) undergo smaller changes in their own lability degree, but these changes have the greater impact on the overall metal flux. Partially labile complexes are more easily influenced by the mixture than labile or inert ones. Some mixture effects can be qualitatively predicted by an analytical expression for the lability index derived using the reaction layer approximation. For a mixture of many complexes, the change in the lability degree of a complex due to the mixture effect can be understood as a combination of the changes due to all of the complexes present.
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The in vitro reactivation of unfolded Escherichia coli alkaline phosphatase (AP) in the presence of the two natively bound metals Zn2+ and Mg2+ produces two protein species, characterized by different guanidine hydrochloride denaturation kinetics. The high-lability AP form slowly converts to the low-lability form in a first-order reaction with a characteristic lifetime (inverse rate constant) of approximately 300 h at pH 8.0 and 25 °C. Addition of Zn2+ and Mg2+ ligands to (folded) apo-AP also produces two protein species, with denaturation kinetics and a long conversion lifetime similar to those found in refolding AP. In contrast, adding Zn2+ alone to apo-AP produces only the high-lability species with no subsequent structural change, suggesting that Mg2+ binding is the event which is responsible for the production of the low-lability AP. The rate of conversion from high- to low-lability AP was found to be linearly dependent on Mg2+ concentration, indicating that Mg2+ binding is rate limiting for this reaction. Experiments where either Zn2+ or Mg2+ was added first, with the second metal added later, show that Mg2+ binding is slowed by the prior presence of bound Zn2+. Mg2+ binding to Zn-AP also slightly increases the enzymatic activity; however, the extent of formation of the low-lability species is related to the square of the Mg2+-induced activity increase. Thus the binding of two Mg2+ to AP produces the dramatic reduction in the rate of denaturation that characterizes the low-lability species. The data suggest the possibility of long distance intersubunit interactions and a role for Mg2+ in providing "kinetic stability" for AP.
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Denaturation (fissile materials)
Guanidine
Reactivity
Enzyme Kinetics
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Lability
Stripping (fiber)
Limiting
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Metabolic stability
Human liver
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The stability of fluoroalkyl groups as a pendent on the phenyl ring was measured in vitro using rat hepatic microsomes and human serum to predict their in vivo stabilities. We have prepared three [$^{18}F$ ]fluoroalkyl-biphenyls as the model compounds of fluoroalkyl aromatic compounds to compare the in vitro stabilities. In addition, in vitro stabilities were measured separately using rat hepatic microsomes and human serum at $37^{\circ}C$ . Fluoroethylbiphenyl had similar or slightly superior stability to fluoropropylbiphenyl and these two compounds were much more stable than fluoromethylbiphenyl in vitro.
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