Preclinical evaluation of WVE-004, aninvestigational stereopure oligonucleotide forthe treatment of C9orf72-associated ALS or FTD.

A large hexanucleotide (G4C2) repeat expansion in the first intronic region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several mechanisms have been proposed to explain how the repeat expansion drives disease, and we hypothesize that a variant-selective approach, in which transcripts affected by the repeat expansion are preferentially decreased, has the potential to address most of them. We report a stereopure antisense oligonucleotide, WVE-004, that executes this variant-selective mechanism of action. WVE-004 dose-dependently and selectively reduces repeat-containing transcripts in patient-derived motor neurons carrying a C9orf72-repeat expansion, as well as in the spinal cord and cortex of C9 BAC transgenic mice. In mice, selective transcript knockdown was accompanied by substantial decreases in dipeptide-repeat proteins, which are pathological biomarkers associated with the repeat expansion, and by preservation of healthy C9orf72 protein expression. These in vivo effects were durable, persisting for at least 6 months. These data support the advancement of WVE-004 as an investigational stereopure antisense oligonucleotide targeting C9orf72 for the treatment of C9orf72-associated ALS or FTD.

Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.

Altered Expression of Transporters, its Potential Mechanisms and Influences in the Liver of Rodent Models Associated with Diabetes Mellitus and Obesity.

Diabetes mellitus is becoming an increasingly prevalent disease that concerns patients and healthcare professionals worldwide. Among many anti-diabetic agents in clinical uses, numerous reports are available on their altered pharmacokinetics because of changes in the expression of drug transporters and metabolic enzymes under diabetic states. These changes may affect the safety and efficacy of therapeutic agents and/or drug-drug interaction with co-administered agents. Therefore, the changes in transporter expression should be identified, and the underlying mechanisms should be clarified. This review summarizes the progress of recent studies on the alterations in important uptake and efflux transporters in liver of diabetic animals and their regulatory pathways.

Genetical study of mutation in maternal-fetal ABO incompatibility.

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother's erythrocytes were agglutinable with monoclonal anti-A antibody (2+) and had agglutination reaction with anti-B antibody (4+). The mother's serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother's erythrocytes yielded agglutination reaction with humanized anti-B serum (4+) and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother's allelic gene was type B and contained type A. The father's blood type was type B, and son's blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01 (ntA640→G)/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Imidazolopiperazines: lead optimization of the second-generation antimalarial agents.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Bid-induced mitochondrial membrane permeabilization waves propagated by local reactive oxygen species (ROS) signaling.

Bid-induced mitochondrial membrane permeabilization and cytochrome c release are central to apoptosis. It remains a mystery how tiny amounts of Bid synchronize the function of a large number of discrete organelles, particularly in mitochondria-rich cells. Looking at cell populations, the rate and lag time of the Bid-induced permeabilization are dose-dependent, but even very low doses lead eventually to complete cytochrome c release. By contrast, individual mitochondria display relatively rapid and uniform kinetics, indicating that the dose dependence seen in populations is due to a spreading of individual events in time. We report that Bid-induced permeabilization and cytochrome c release regularly demonstrate a wave-like pattern, propagating through a cell at a constant velocity without dissipation. Such waves do not depend on caspase activation or permeability transition pore opening. However, reactive oxygen species (ROS) scavengers suppressed the coordination of cytochrome c release and also inhibited Bid-induced cell death, whereas both superoxide and hydrogen peroxide sensitized mitochondria to Bid-induced permeabilization. Thus, Bid engages a ROS-dependent, local intermitochondrial potentiation mechanism that amplifies the apoptotic signal as a wave.

Imidazolopiperazines: hit to lead optimization of new antimalarial agents.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

Attenuation of intestinal absorption by major efflux transporters: quantitative tools and strategies using a Caco-2 model.

Efflux transporters expressed in the apical membrane of intestinal enterocytes have been implicated in drug oral absorption. The current study presents a strategy and tools to quantitatively predict the impact of efflux on oral absorption for new chemical entities (NCEs) in early drug discovery. Sixty-three marketed drugs with human absorption data were evaluated in the Caco-2 bidirectional permeability assay and subjected to specific transporter inhibition. A four-zone graphical model was developed from apparent permeability and efflux ratios to quickly identify compounds whose efflux activity may distinctly influence human absorption. NCEs in "zone 4" will probably have efflux as a barrier for oral absorption and further mechanistic studies are required. To interpret mechanistic results, we introduced a new quantitative substrate classification parameter, transporter substrate index (TSI). TSI allowed more flexibility and considered both in vitro and in vivo outcomes. Its application ranged from addressing the challenge of overlapping substrate specificity to projecting the role of transporter(s) on exposure or potential drug-drug interaction risk. The potential impact of efflux transporters associated with physicochemical properties on drug absorption is discussed in the context of TSI and also the previously reported absorption quotient. In this way, the chemistry strategy may be differentially focused on passive permeability or efflux activity or both.

Towards prediction of in vivo intestinal absorption using a 96-well Caco-2 assay.

We systematically validated a robust 96-well Caco-2 assay via an extended set of 93 marketed drugs with diverse transport mechanisms and quantified by LC/MS/MS, to investigate its predictive utility while dealing with challenging discovery compounds. Utilizing nonlinear fit, the validation led to a good correlation (R(2) = 0.76) between absorptive permeability, log P(app)(A-B), from in vitro Caco-2 assay and reported human fraction of dose absorbed. We observed that paracellular compounds could be flagged by log P(app)(A-B) (<-5.5 cm/s) and physicochemical property space (c log P < 1). Of 8000 Novartis discovery compounds examined 13% were subject to low recovery (<30%). Compound loss was investigated by comparing cell monolayer and artificial membrane, while 0.5% bovine serum albumin (in both donor and acceptor compartments) was utilized to improve recovery. The second focus of this study was to investigate the advantages and limitations of the current Caco-2 assay for predicting in vivo intestinal absorption. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with suboptimal physicochemical properties, Caco-2 data successfully projected low intestinal absorption. This platform sets the stage for mechanistically evaluating compounds towards improving in vitro-in vivo correlations.

Control of calcium signal propagation to the mitochondria by inositol 1,4,5-trisphosphate-binding proteins.

Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224-605). As expected, p130PH-GFP and GFP-IP3R224-605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake.

Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells.

Neuropilin-1 (NP1), in conjunction with plexins, promotes axon repulsion by binding to semaphorin 3A (SEMA3A). Although NP1 is expressed in carcinoma cells, its functions have remained elusive, and neither SEMA3A nor plexin expression has been explored in cancer. Here we provide evidence that breast carcinoma cells support an autocrine pathway involving SEMA3A, plexin-A1, and NP1 that impedes their ability to chemotax. Reducing SEMA3A or NP1 expression by RNA interference or inhibiting plexin-A1 signaling enhanced migration. Conversely, expression of constitutively active plexin-A1 impaired chemotaxis. The paradox of how breast carcinoma cells expressing these endogenous chemotaxis inhibitors are able to migrate is explained by their expression of vascular endothelial growth factor (VEGF), a NP1 ligand that competes with SEMA3A for receptor binding. Finally, we establish that the ratio of endogenous VEGF and SEMA3A concentrations in carcinoma cells determines their chemotactic rate. Our findings lead to the surprising conclusion that opposing autocrine loops involving NP1 regulate the chemotaxis of breast carcinoma cells. Moreover, our data indicate a novel autocrine function for VEGF in chemotaxis.

Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130.

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.