Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants

ABSTRACT Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2,4-diamino-pyrrolo [2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer. A series of novel 2,4-diamino-pyrrolo[2,3-d]pyrimidines were described as potent inhibitors of iron-dependent lipid peroxidation, and proved to be cytoprotective in cell culture models of oxidative injury and neuroprotective in brain injury and ischemia models Structural determinants of permeability and partitioning are discussed for a series of structurally similar homologs. In addition, detailed studies were conducted concurrently with two radiolabled compounds from the pyrrolopyrimidine series representing different physicochemical, permeability, and cell partitioning attributes to discern the roles of protein binding and cell partitioning on permeation and to complement ongoing pharmacological and pharmacokinetic studies. The data proved useful in predicting which compounds were most likely to leave the blood and penetrate underlying tissue. In a companion paper, brain uptake dynamics and cellular penetration of these compounds are confirmed in viv

Integration of in Silico and in Vitro Tools for Scaffold Optimization during Drug Discovery: Predicting P‑Glycoprotein Efflux

In silico tools are regularly utilized for designing and prioritizing compounds to address challenges related to drug metabolism and pharmacokinetics (DMPK) during the process of drug discovery. P-Glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) transporters with broad substrate specificity that plays a significant role in absorption and distribution of drugs that are P-gp substrates. As a result, screening for P-gp transport has now become routine in the drug discovery process. Typically, bidirectional permeability assays are employed to assess in vitro P-gp efflux. In this article, we use P-gp as an example to illustrate a well-validated methodology to effectively integrate in silico and in vitro tools to identify and resolve key barriers during the early stages of drug discovery. A detailed account of development and application of in silico tools such as simple guidelines based on physicochemical properties and more complex quantitative structure–activity relationship (QSAR) models is provided. The tools were developed based on structurally diverse data for more than 2000 compounds generated using a robust P-gp substrate assay over the past several years. Analysis of physicochemical properties revealed a significantly lower proportion (<10%) of P-gp substrates among the compounds with topological polar surface area (TPSA) <60 Ų and the most basic cpKa <8. In contrast, this proportion of substrates was greater than 75% for compounds with TPSA >60 Ų and the most basic cpKa >8. Among the various QSAR models evaluated to predict P-gp efflux, the Bagging model provided optimum prediction performance for prospective validation based on chronological test sets. Four sequential versions of the model were built with increasing numbers of compounds to train the models as new data became available. Except for the first version with the smallest training set, the QSAR models exhibited robust prediction profiles with positive prediction values (PPV) and negative prediction values (NPV) exceeding 80%. The QSAR model demonstrated better concordance with the manual P-gp substrate assay than an automated P-gp substrate screen. The in silico and the in vitro tools have been effectively integrated during early stages of drug discovery to resolve P-gp-related challenges exemplified by several case studies. Key learning based on our experience with P-gp can be widely applicable across other DMPK-related challenges

In Silico and in Vitro Assessment of OATP1B1 Inhibition in Drug Discovery

The organic anion-transporting polypeptide 1B1 transporter belongs to the solute carrier superfamily and is highly expressed at the basolateral membrane of hepatocytes. Several clinical studies show drug–drug interactions involving OATP1B1, thereby prompting the International Transporter Consortium to label OATP1B1 as a critical transporter that can influence a compound’s disposition. To examine OATP1B1 inhibition early in the drug discovery process, we established a medium-throughput concentration-dependent OATP1B1 assay. To create an in silico OATP1B1 inhibition model, deliberate in vitro assay enrichment was performed with publically known OATP1B1 inhibitors, noninhibitors, and compounds from our own internal chemistry. To date, approximately 1200 compounds have been tested in the assay with 60:40 distribution between noninhibitors and inhibitors. Bagging, random forest, and support vector machine fingerprint (SVM-FP) quantitative structure–activity relationship classification models were created, and each method showed positive and negative predictive values >90%, sensitivity >80%, specificity >95%, and Matthews correlation coefficient >0.8 on a prospective test set indicating the ability to distinguish inhibitors from noninhibitors. A SVMF-FP regression model was also created that showed an <i>R</i>² of 0.39, Spearman’s rho equal to 0.76, and was capable of predicting 69% of the prospective test set within the experimental variability of the assay (3-fold). In addition to the in silico quantitative structure–activity relationship (QSAR) models, physicochemical trends were examined to provide structure activity relationship guidance to early discovery teams. A JMP partition tree analysis showed that among the compounds with calculated logP >3.5 and ≥1 negatively charged atom, 94% were identified as OATP1B1 inhibitors. The combination of the physicochemical trends along with an in silico QSAR model provides discovery project teams a valuable tool to identify and address drug–drug interaction liability due to OATP1B1 inhibition

Breast Cancer Resistance Protein Interacts with Various Compounds in Vitro, but Plays a Minor Role in Substrate Efflux at the Blood-Brain Barrier

Expression of breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) has been revealed recently. To investigate comprehensively the potential role of Bcrp at the murine BBB, a chemically diverse set of model compounds (cimetidine, alfuzosin, dipyridamole, and LY2228820) was evaluated using a multiexperimental design. Bcrp1 stably transfected MDCKII cell monolayer transport studies demonstrated that each compound had affinity for Bcrp and that polarized transport by Bcrp was abolished completely by the Bcrp inhibitor chrysin. However, none of the compounds differed in brain uptake between Bcrp wild-type and knockout mice under either an in situ brain perfusion or a 24-h subcutaneous osmotic minipump continuous infusion experimental paradigm. In addition, alfuzosin and dipyridamole were shown to undergo transport by P-glycoprotein (P-gp) in an MDCKII-MDR1 cell monolayer model. Alfuzosin brain uptake was 4-fold higher in mdr1a(–/–) mice than in mdr1a(+/+) mice in in situ and in vivo studies, demonstrating for the first time that it undergoes P-gp-mediated efflux at the BBB. In contrast, P-gp had no effect on dipyridamole brain penetration in situ or in vivo. In fact, in situ BBB permeability of these solutes appeared to be primarily dependent on their lipophilicity in the absence of efflux transport, and in situ brain uptake clearance correlated with the intrinsic transcellular passive permeability from in vitro transport and cellular accumulation studies. In summary, Bcrp mediates in vitro transport of various compounds, but seems to play a minimal role at the BBB in vivo

Selenorhodamine Photosensitizers for Photodynamic Therapy of P‑Glycoprotein-Expressing Cancer Cells

We examined a series of selenorhodamines with amide and thioamide functionality at the 5-position of a 9-(2-thienyl) substituent on the selenorhodamine core for their potential as photosensitizers for photodynamic therapy (PDT) in P-glycoprotein (P-gp) expressing cells. These compounds were examined for their photophysical properties (absorption, fluorescence, and ability to generate singlet oxygen), for their uptake into Colo-26 cells in the absence or presence of verapamil, for their dark and phototoxicity toward Colo-26 cells, for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their colocalization with mitochondrial specific agents in Colo-26 cells. Thioamide derivatives <b>16b</b> and <b>18b</b> were more effective photosensitizers than amide derivatives <b>15b</b> and <b>17b</b>. Selenorhodamine thioamides <b>16b</b> and <b>18b</b> were useful in a combination therapy to treat Colo-26 cells in vitro: a synergistic therapeutic effect was observed when Colo-26 cells were exposed to PDT and treatment with the cancer drug doxorubicin

Stereochemistry Balances Cell Permeability and Solubility in the Naturally Derived Phepropeptin Cyclic Peptides

Cyclic peptide (CP) natural products provide useful model systems for mapping “beyond-Rule-of-5” (bRo5) space. We identified the phepropeptins as natural product CPs with potential cell permeability. Synthesis of the phepropeptins and epimeric analogues revealed much more rapid cellular permeability for the natural stereochemical pattern. Despite being more cell permeable, the natural compounds exhibited similar aqueous solubility as the corresponding epimers, a phenomenon explained by solvent-dependent conformational flexibility among the natural compounds. When analyzing the polarity of the solution structures we found that neither the number of hydrogen bonds nor the total polar surface area accurately represents the solvation energies of the high and low dielectric conformations. This work adds to a growing number of natural CPs whose solvent-dependent conformational behavior allows for a balance between aqueous solubility and cell permeability, highlighting structural flexibility as an important consideration in the design of molecules in bRo5 chemical space