Chronic treatment with asenapine affects cytochrome P450 2D (CYP2D) in rat brain and liver. Pharmacological aspects.

Neuroleptics have to be used for a long time to produce a therapeutic effect. Cytochrome P450 2D (CYP2D) enzymes mediate alternative pathways of neurotransmitter synthesis (i.e. tyramine hydroxylation to dopamine and 5-methoxytryptamine O-demethylation to serotonin), and metabolism of neurosteroids. The aim of our present study was to examine the influence of chronic treatment with the new atypical neuroleptic asenapine on CYP2D in rat brain. In parallel, liver CYP2D was investigated for comparison. Asenapine added in vitro to microsomes of control rats competitively, but weakly inhibited the activity of CYP2D (brain: Ki = 385 µM; liver: Ki = 36 µM). However, prolonged administration of asenapine (0.3 mg/kg sc. for 2 weeks) significantly diminished the activity and protein level of CYP2D in the frontal cortex, nucleus accumbens, hippocampus and cerebellum, but did not affect the enzyme in the hypothalamus, brain stem, substantia nigra and the remainder of the brain. In contrast, asenapine enhanced the enzyme activity and protein level in the striatum. In the liver, chronically administered asenapine reduced the activity and protein level of CYP2D, and the CYP2D1 mRNA level. In conclusion, prolonged administration of asenapine alters the CYP2D expression in the brain structures and in the liver. Through affecting the CYP2D activity in the brain, asenapine may modify its pharmacological effect. By increasing the CYP2D expression/activity in the striatum, asenapine may accelerate the synthesis of dopamine (via tyramine hydroxylation) and serotonin (via 5-methoxytryptamine O-demethylation), and thus alleviate extrapyramidal symptoms. By reducing the CYP2D expression/activity in other brain structures asenapine may diminish the 21-hydroxylation of neurosteroids and thus have a beneficial influence on the symptoms of schizophrenia. In the liver, by reducing the CYP2D activity, asenapine may slow the biotransformation of concomitantly administered CYP2D substrates (drugs) during continuous treatment of schizophrenia or bipolar disorders.

Asenapine maleate inhibits angiotensin II-induced proliferation and activation of cardiac fibroblasts via the ROS/TGFß1/MAPK signaling pathway.

BACKGROUND: Cardiac fibrosis will increase wall stiffness and diastolic dysfunction, which will eventually lead to heart failure. Asenapine maleate (AM) is widely used in the treatment of schizophrenia. In the current study, we explored the potential mechanism underlying the role of AM in angiotensin II (Ang II)-induced cardiac fibrosis. METHODS: Cardiac fibroblasts (CFs) were stimulated using Ang II with or without AM. Cell proliferation was measured using the cell counting kit-8 assay and the Cell-Light EdU Apollo567 In Vitro Kit. The expression levels of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were detected using immunofluorescence or western blotting. At the protein level, the expression levels of the components of the transforming growth factor beta 1 (TGFß1)/mitogen-activated protein kinase (MAPK) signaling pathway were also detected. RESULTS: After Ang II stimulation, TGFß1, TGFß1 receptor, α-SMA, fibronectin (Fn), collagen type I (Col1), and collagen type III (Col3) mRNA levels increased; the TGFß1/MAPK signaling pathway was activated in CFs. After AM pretreatment, cell proliferation was inhibited, the numbers of PCNA -positive cells and the levels of cardiac fibrosis markers decreased. The activity of the TGFß1/MAPK signaling pathway was also inhibited. Therefore, AM can inhibit cardiac fibrosis by blocking the Ang II-induced activation through TGFß1/MAPK signaling pathway. CONCLUSIONS: This is the first report to demonstrate that AM can inhibit Ang II-induced cardiac fibrosis by down-regulating the TGFß1/MAPK signaling pathway. In this process, AM inhibited the proliferation and activation of CFs and reduced the levels of cardiac fibrosis markers. Thus, AM represents a potential treatment strategy for cardiac fibrosis.

Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Chronic antipsychotic treatment exerts limited effects on the mania-like behavior of dopamine transporter knockdown mice.

BACKGROUND: Bipolar disorder is a life-threatening disorder linked to dopamine transporter (DAT) polymorphisms, with reduced DAT levels seen in positron emission tomography and postmortem brains. AIMS: The purpose of this study was to examine the effects of approved antipsychotics on DAT dysfunction-mediated mania behavior in mice. METHODS: DAT knockdown mice received either D2-family receptor antagonist risperidone or asenapine and mania-related behaviors were assessed in the clinically-relevant behavioral pattern monitor to assess spontaneous exploration. RESULTS: Chronic risperidone did not reverse mania-like behavior in DAT knockdown mice. Chronic asenapine reduced mania behavior but this effect was more pronounced in wild-type littermates than in DAT knockdown mice. CONCLUSION: Taken together, these findings suggest that while acute antipsychotic treatment may be beneficial in management of bipolar mania, more targeted therapeutics may be necessary for long-term treatment. Specific investigation into DAT-targeting drugs could improve future treatment of bipolar mania.

Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Impact of P-Glycoprotein-Mediated Active Efflux on Drug Distribution into Lumbar Cerebrospinal Fluid in Nonhuman Primates.

Estimation of unbound drug concentration in the brain (Cu,brain) is an essential part of central nervous system (CNS) drug development. As a surrogate for Cu,brain in humans and nonhuman primates, drug concentration in cerebrospinal fluid (CCSF) collected by lumbar puncture is often used; however, the predictability of Cu,brain by lumbar CCSF is unclear, particularly for substrates of the active efflux transporter P-glycoprotein (P-gp). Here, we measured lumbar CCSF in cynomolgus monkey after single intravenous administration of 10 test compounds with varying P-gp transport activities. The in vivo lumbar cerebrospinal fluid (CSF)-to-plasma unbound drug concentration ratios (Kp,uu,lumbar CSF) of nonsubstrates or weak substrates of P-gp were in the range 0.885-1.34, whereas those of good substrates of P-gp were in the range 0.195-0.458 and were strongly negatively correlated with in vitro P-gp transport activity. Moreover, concomitant treatment with a P-gp inhibitor, zosuquidar, increased the Kp,uu,lumbar CSF values of the good P-gp substrates, indicating that P-gp-mediated active efflux contributed to the low Kp,uu,lumbar CSF values of these compounds. Compared with the drug concentrations in the cisternal CSF and interstitial fluid (ISF) that we previously determined in cynomolgus monkeys, the lumbar CCSF were more than triple for two and all of the good P-gp substrates examined, respectively. Although lumbar CCSF may overestimate cisternal CSF and ISF concentrations of good P-gp substrates, lumbar CCSF allowed discrimination of good P-gp substrates from the weak and nonsubstrates and can be used to estimate the impact of P-gp-mediated active efflux on drug CNS penetration. SIGNIFICANCE STATEMENT: This is the first study to systematically evaluate the penetration of various P-glycoprotein (P-gp) substrates into lumbar cerebrospinal fluid (CSF) in nonhuman primates. Lumbar CSF may contain >3-fold higher concentrations of good P-gp substrates than interstitial fluid (ISF) and cisternal CSF but was able to discriminate the good substrates from the weak or nonsubstrates. Because lumbar CSF is more accessible than ISF and cisternal CSF in nonhuman primates, these findings will help increase our understanding of drug central nervous system penetration at the nonclinical stage.

Elucidation of cGMP-dependent induction of mitochondrial biogenesis through PKG and p38 MAPK in the kidney.

Previous studies have shown that cGMP increases mitochondrial biogenesis (MB). Our laboratory has determined that formoterol and LY344864, agonists of the ß2-adrenergic receptor and 5-HT1F receptor, respectively, signal MB in a soluble guanylyl cyclase (sGC)-dependent manner. However, the pathway between cGMP and MB produced by these pharmacological agents in renal proximal tubule cells (RPTCs) and the kidney has not been determined. In the present study, we showed that treatment of RPTCs with formoterol, LY344864, or riociguat, a sGC stimulator, induces MB through protein kinase G (PKG), a target of cGMP, and p38, an associated downstream target of PKG and a regulator of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in RPTCs. We also examined if p38 plays a role in PGC-1α phosphorylation in vivo. Administration of l-skepinone, a potent and specific inhibitor of p38α and p38ß, to naïve mice inhibited phosphorylated PGC-1α localization in the nuclear fraction of the renal cortex. Taken together, we demonstrated a pathway, sGC/cGMP/PKG/p38/PGC-1α, for pharmacological induction of MB and the importance of p38 in this pathway.

A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2.

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.

ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators.

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37-80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors.

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5'-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.

Evaluation of the therapeutic potential of the selective p38 MAPK inhibitor Skepinone-L and the dual p38/JNK 3 inhibitor LN 950 in experimental K/BxN serum transfer arthritis.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling plays an important role in inflammatory diseases such as rheumatoid arthritis (RA).The aim of our study was to elucidate the therapeutic potential of the highly selective p38 MAPK inhibitor Skepinone-L and the dual inhibitor LN 950 (p38 MAPK and JNK 3) in the K/BxN serum transfer model of RA. Additionally, we aimed to monitor MAPK treatment non-invasively in vivo using the hypoxia tracer [18F]fluoromisonidazole ([18F]FMISO) and positron emission tomography (PET). METHODS: To induce experimental arthritis, we injected glucose-6-phosphate isomerase autoantibody-containing serum in BALB/c mice. MAPK inhibitor or Sham treatment was administered per os once daily. On days 3 and 6 after arthritis induction, we conducted PET imaging with [18F]FMISO. At the end of the experiment, ankles were harvested for histopathological analysis. RESULTS: Skepinone-L and LN 950 were applicable to suppress the severity of experimental arthritis confirmed by reduced ankle swelling and histopathological analysis. Skepinone-L (3.18 ± 0.19 mm) and LN 950 (3.40 ± 0.13 mm) treatment yielded a significantly reduced ankle thickness compared to Sham-treated mice (3.62 ± 0.11 mm) on day 5 after autoantibody transfer, a time-point characterized by severe arthritis. Hypoxia imaging with [18F]FMISO revealed non-conclusive results and might not be an appropriate tool to monitor MAPK therapy in experimental RA. CONCLUSION: Both the selective p38 MAPK inhibitor Skepinone-L and the dual (p38 MAPK and JNK 3) inhibitor LN 950 exhibited significant therapeutic effects during experimental arthritis. Thus, our study contributes to the ongoing discussion on the use of p38 MAPK as a potential target in RA.

Intracellular Doppler Spectroscopy detects altered drug response in SKOV3 tumor spheroids with silenced or inhibited P-glycoprotein.

Intracellular Doppler spectroscopy is a form of low-coherence digital holography based upon Doppler detection of scattered light that measures drug response/resistance in tumor spheroids, xenografts, and clinical biopsies. Multidrug resistance (MDR) is one of the main causes of ineffective cancer treatment. One MDR mechanism is mediated by the MDR1 gene that encodes the drug efflux pump P-glycoprotein (Pgp). Overexpression of Pgp in some cancers is associated with poor chemotherapeutic response. This paper uses intracellular Doppler spectroscopy to detect Pgp-mediated changes to drug response in 3D tissues grown from an ovarian cancer cell line (SKOV3). The SKOV3 cell line was incrementally exposed to cisplatin to create a cell line with increased Pgp expression (SKOV3cis). Subsequently, MDR1 in a subset of these cells was silenced in SKOV3cis using shRNA to create a doxycycline inducible, Pgp-silenced cell line (SKOV3cis-sh). A specific Pgp inhibitor, zosuquidar, was used to study the effects of Pgp inhibition on the Doppler spectra. Increased drug sensitivity was observed with Pgp silencing or inhibition as determined by drug IC50s of paclitaxel-response of silenced Pgp and doxorubicin-response of inhibited Pgp, respectively. These results indicate that intracellular Doppler spectroscopy can detect changes in drug response due to silencing or inhibition of a single protein associated with drug resistance with important consequences for personalized medicine.

Structural insight into substrate and inhibitor discrimination by human P-glycoprotein.

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo-electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate-hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

Evidence of a role for functional heterogeneity in multidrug resistance transporters in clinical trials of P-glycoprotein modulation in acute myeloid leukemia.

BACKGROUND: Multidrug resistance (MDR) transporter proteins such as P-glycoprotein (P-gp) efflux a variety of chemotherapeutic drugs from acute myeloid leukemia (AML) blasts leading to clinical drug resistance. METHODS: This study examined heterogeneity of MDR functional efflux by AML blasts using two flow cytometry bioassays. Bone marrow specimens (N = 50) from elderly patients with newly diagnosed AML were analyzed for CD34+ blasts with MDR efflux function. Efflux was measured with a fluorescent dye (DiOC2 ) as a surrogate for oncology drugs that are substrates for MDR efflux. P-gp-mediated efflux was differentiated from non-P-gp MDR activities using zosuquidar, a highly selective P-gp modulator. The bioassays included a zosuquidar-dependent DiOC2 accumulation bioassay that measured only P-gp. The second method, termed the efflux bioassay, could detect P-gp and other non-P-gp efflux depending on bioassay culture conditions. RESULTS: Sixty-two percent of the specimens were considered positive for blasts with P-gp function, and 26% of such P-gp-positive specimens also exhibited zosuquidar-resistant (i.e., non-P-gp) MDR efflux activity; 37% of P-gp-negative AML blast specimens displayed zosuquidar-resistant MDR function in the efflux bioassay. CONCLUSIONS: These results confirm the heterogeneous nature of MDR efflux pumps in AML blasts, and provide support for the hypothesis that non-P-gp MDR contributed to negative results with zosuquidar in AML trials like ECOG-ACRIN E3999. © 2018 International Clinical Cytometry Society.

Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y2 Receptor Based on AR-C118925.

The human P2Y2 receptor ( hP2Y2R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y2R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y2R antagonists, based on the known, non-nucleotide hP2Y2R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y2R (p Kd = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y2R. These properties make 98 one of the first tools for studying hP2Y2R distribution and organization.

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes.

BACKGROUND: TGFß1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFß1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved. METHODS: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. RESULTS: TGFß1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFß1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFß1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). CONCLUSIONS: TGFß1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.

Abcb1a (P-glycoprotein) limits brain exposure of the anticancer drug candidate seliciclib in vivo in adult mice.

Seliciclib displayed limited brain exposure in vivo in adult rats with mature blood-brain barrier (BBB). Selicilib was shown to be a specific substrate of human ABCB1 in vitro. To demonstrate that ABCB1/Abcb1 can limit brain exposure in vivo in mice we are showing that seliciclib is a substrate of mouse Abcb1a, the murine ABCB1 ortholog expressed in the BBB as LLC-PK-Abcb1a cells displayed an efflux ratio (ER) of 15.31±3.54 versus an ER of 1.44±0.10 in LLC-PK1-mock cells. Additionally, in the presence of LY335979, an ABCB1/Abcb1a specific inhibitor, the observed ER for seliciclib in the LLC-PK1-mMdr1a cells decreased to 1.05±0.25. To demonstrate in vivo relevance of seliciclib transport by Abcb1a mouse brain microdialysis experiments were carried out that showed that the AUCbrain/AUCblood ratio of 0.143 in anesthetized mice increased about two-fold to 0.279 in the presence of PSC833 another ABCB1/Abcb1a specific inhibitor. PSC833 also increased the brain exposure (AUCbrain) of seliciclib close to 2-fold (136 vs 242) in awake mice. In sum, Abcb1a significantly decreases seliciclib permeability in vitro and is partly responsible for limited brain exposure of seliciclib in vivo in mice.

A novel individual-cell-based mathematical model based on multicellular tumour spheroids for evaluating doxorubicin-related delivery in avascular regions.

BACKGROUND AND PURPOSE: Effective drug delivery in the avascular regions of tumours, which is crucial for the promising antitumour activity of doxorubicin-related therapy, is governed by two inseparable processes: intercellular diffusion and intracellular retention. To accurately evaluate doxorubicin-related delivery in the avascular regions, these two processes should be assessed together. Here we describe a new approach to such an assessment. EXPERIMENTAL APPROACH: An individual-cell-based mathematical model based on multicellular tumour spheroids was developed that describes the different intercellular diffusion and intracellular retention kinetics of doxorubicin in each cell layer. The different effects of a P-glycoprotein inhibitor (LY335979) and a hypoxia inhibitor (YC-1) were quantitatively evaluated and compared, in vitro (tumour spheroids) and in vivo (HepG2 tumours in mice). This approach was further tested by evaluating in these models, an experimental doxorubicin derivative, INNO 206, which is in Phase II clinical trials. KEY RESULTS: Inhomogeneous, hypoxia-induced, P-glycoprotein expression compromised active transport of doxorubicin in the central area, that is, far from the vasculature. LY335979 inhibited efflux due to P-glycoprotein but limited levels of doxorubicin outside the inner cells, whereas YC-1 co-administration specifically increased doxorubicin accumulation in the inner cells without affecting the extracellular levels. INNO 206 exhibited a more effective distribution profile than doxorubicin. CONCLUSIONS AND IMPLICATIONS: The individual-cell-based mathematical model accurately evaluated and predicted doxorubicin-related delivery and regulation in the avascular regions of tumours. The described framework provides a mechanistic basis for the proper development of doxorubicin-related drug co-administration profiles and nanoparticle development and could avoid unnecessary clinical trials.

Optimized Target Residence Time: Type†I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography.

Reversion of P-gp-Mediated Drug Resistance in Ovarian Carcinoma Cells with PHPMA-Zosuquidar Conjugates.

Inhibition of P-glycoprotein (P-gp) transporter is an attractive approach for the reversion of cancer-associated multidrug resistance (MDR). Poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA)-based carriers that are able to release the anticancer drug doxorubicin in the lysosomes have shown promise to reduce P-gp mediated resistance. This is attributed to the release of the drug in close proximity to the nucleus and distant from the P-gp transporter. This work presents a strategy to maximize P-gp inhibition and enhance doxorubicin cytotoxicity in cancer cells by using a dual functional PHPMA conjugate carrying both the anticancer drug doxorubicin and the P-glycoprotein inhibitor zosuquidar (Zos). While doxorubicin was connected to the polymer backbone via a lysosomally cleavable spacer, the P-gp inhibitor Zos was attached by a hydrazone linker in order to promote release in the early stage of the endocytic process and maximize its cytosolic concentration in proximity of the P-gp transporter. Following Zos modification and determination of its ability to inhibit P-gp, conjugation to the PHPMA polymer backbone resulted in enhanced doxorubicin cytotoxicity in resistant A2780ADR ovarian carcinoma cells. Finally, the incorporation of both Dox and Zos in a single polymer carrier enhanced P-gp inhibition as compared to a control PHPMA conjugate containing only Dox.