Publications by authors named "Curt Mazur"

30 Publications

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The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration.

Nucleic Acids Res 2021 01;49(2):657-673

Ionis Pharmaceuticals Inc. Carlsbad, CA 92010, USA.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.
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http://dx.doi.org/10.1093/nar/gkaa1235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826274PMC
January 2021

Convective forces increase rostral delivery of intrathecal radiotracers and antisense oligonucleotides in the cynomolgus monkey nervous system.

J Transl Med 2020 08 8;18(1):309. Epub 2020 Aug 8.

Biogen, Inc., Cambridge, MA, USA.

Background: The intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys.

Methods: There are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer Tc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging.

Results: It was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm.

Conclusions: These results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.
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http://dx.doi.org/10.1186/s12967-020-02461-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414676PMC
August 2020

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System.

J Vis Exp 2019 10 29(152). Epub 2019 Oct 29.

Biogen, Inc.;

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.
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http://dx.doi.org/10.3791/60274DOI Listing
October 2019

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

JCI Insight 2019 10 17;4(20). Epub 2019 Oct 17.

Biogen, Cambridge, Masschusetts, USA.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.
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http://dx.doi.org/10.1172/jci.insight.129240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824309PMC
October 2019

Antisense oligonucleotides extend survival of prion-infected mice.

JCI Insight 2019 07 30;5. Epub 2019 Jul 30.

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, Montana, USA.

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.
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http://dx.doi.org/10.1172/jci.insight.131175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777807PMC
July 2019

A modular analysis of microglia gene expression, insights into the aged phenotype.

BMC Genomics 2019 Feb 28;20(1):164. Epub 2019 Feb 28.

Ionis Pharmaceuticals, Inc, Carlsbad, CA, 92010, USA.

Background: Microglia are multifunctional cells that are key players in brain development and homeostasis. Recent years have seen tremendous growth in our understanding of the role microglia play in neurodegeneration, CNS injury, and developmental disorders. Given that microglia show diverse functional phenotypes, there is a need for more precise tools to characterize microglial states. Here, we experimentally define gene modules as the foundation for describing microglial functional states.

Results: In an effort to develop a comprehensive classification scheme, we profiled transcriptomes of mouse microglia in a stimulus panel with 96 different conditions. Using the transcriptomic data, we generated fine-resolution gene modules that are robustly preserved across datasets. These modules served as the basis for a combinatorial code that we then used to characterize microglial activation under various inflammatory stimulus conditions.

Conclusions: The microglial gene modules described here were robustly preserved, and could be applied to in vivo as well as in vitro conditions to dissociate the signaling pathways that distinguish acutely inflamed microglia from aged microglia. The microglial gene modules presented here are a novel resource for classifying and characterizing microglial states in health and disease.
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http://dx.doi.org/10.1186/s12864-019-5549-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396472PMC
February 2019

Time-dependent, bidirectional, anti- and pro-spinal hyper-reflexia and muscle spasticity effect after chronic spinal glycine transporter 2 (GlyT2) oligonucleotide-induced downregulation.

Exp Neurol 2018 07 30;305:66-75. Epub 2018 Mar 30.

Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, La Jolla, CA, USA; Institute of Neurobiology, Slovak Academy of Sciences, Soltesovej 6, Kosice -04001, Slovak Republic. Electronic address:

The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we characterize the effect of spinally-targeted GlyT2 downregulation once initiated at chronic stages after induction of spasticity in rats. In animals with identified hyper-reflexia, the anti-spasticity effect was studied after intrathecal treatment with: i) glycine, ii) GlyT2 inhibitor (ALX 1393), and iii) GlyT2 antisense oligonucleotide (GlyT2-ASO). Administration of glycine and GlyT2 inhibitor led to significant suppression of spasticity lasting for a minimum of 45-60 min. Treatment with GlyT2-ASO led to progressive suppression of muscle spasticity seen at 2-3 weeks after treatment. Over the subsequent 4-12 weeks, however, the gradual appearance of profound spinal hyper-reflexia was seen. This was presented as spontaneous or slight-tactile stimulus-evoked muscle oscillations in the hind limbs (but not in upper limbs) with individual hyper-reflexive episodes lasting between 3 and 5 min. Chronic hyper-reflexia induced by GlyT2-ASO treatment was effectively blocked by intrathecal glycine. Immunofluorescence staining and Q-PCR analysis of the lumbar spinal cord region showed a significant (>90%) decrease in GlyT2 mRNA and GlyT2 protein. These data demonstrate that spinal GlyT2 downregulation provides only a time-limited therapeutic benefit and that subsequent loss of glycine vesicular synthesis resulting from chronic GlyT2 downregulation near completely eliminates the tonic glycine-ergic activity and is functionally expressed as profound spinal hyper-reflexia. These characteristics also suggest that chronic spinal GlyT2 silencing may be associated with pro-nociceptive activity.
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http://dx.doi.org/10.1016/j.expneurol.2018.03.013DOI Listing
July 2018

Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander disease.

Ann Neurol 2018 01 14;83(1):27-39. Epub 2018 Jan 14.

Waisman Center, University of Wisconsin-Madison, Madison, WI.

Objective: Alexander disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander disease.

Methods: In this study, we use GFAP mutant mouse models of Alexander disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP.

Results: Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis.

Interpretation: These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander disease. Ann Neurol 2018;83:27-39.
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http://dx.doi.org/10.1002/ana.25118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876100PMC
January 2018

Antisense oligonucleotides selectively suppress target RNA in nociceptive neurons of the pain system and can ameliorate mechanical pain.

Pain 2018 01;159(1):139-149

Neuroscience Drug Discovery, Ionis Pharmaceuticals Inc, Carlsbad, CA, USA.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001074DOI Listing
January 2018

Development of a simple, rapid, and robust intrathecal catheterization method in the rat.

J Neurosci Methods 2017 03 2;280:36-46. Epub 2017 Feb 2.

Ionis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA.

Background: The blood brain barrier (BBB) is an impediment to the development of large and highly charged molecules as therapeutics for diseases and injuries of the central nervous system (CNS). Antisense oligonucleotides (ASOs) are large (6000-8000MW) and highly charged and therefore do not cross the BBB. A method of circumventing the blood brain barrier to test ASOs, and other non-BBB penetrant molecules, as CNS therapeutics is the direct administration of these molecules to the CNS tissue or cerebral spinal fluid.

New Method: We developed a rapid, simple and robust method for the intrathecal catheterization of rats to test putatively therapeutic antisense oligonucleotides. This method utilizes 23-gauge needles, simply constructed ½in. long 19-gauge guide cannulas and 8cm long plastic PE-10 sized catheters.

Comparison With Existing Methods: Unlike the cisterna magna approach, this method uses a lumbar approach for intrathecal catheterization with the catheter residing entirely in the cauda equina space minimizing spinal cord compression. Readily available materials and only a few specialized pieces of equipment, which are easily manufactured, are used for this intrathecal catheterization method.

Conclusions: This method is easy to learn and has been taught to multiple in house surgeons, collaborators and contract laboratories. Greater than 90% catheterization success is routinely achieved with this method and as many as 100 catheters can be placed and test substance administered in one 6-h period. This method has allowed the pre-clinical testing of hundreds of ASOs as therapeutics for CNS indications.
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http://dx.doi.org/10.1016/j.jneumeth.2017.02.001DOI Listing
March 2017

Identification of REST-regulated genes and pathways using a REST-targeted antisense approach.

Nucleic Acid Ther 2013 Dec;23(6):389-400

1 Department of Molecular Neurobiology, Evotec AG , Hamburg, Germany .

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is one of the first negative-acting transcriptional regulators implicated in vertebrate development thought to regulate hundreds of neuron-specific genes. However, its function in the adult system remains elusive. Here we employ second-generation antisense oligonucleotides (ASOs) to study the impact of rest-mediated suppression on gene expression. We demonstrate specific reductions in REST levels in vitro, and in vivo in mouse liver following treatment with ASOs, and we show that ASO mediated-REST suppression results in the elevation in expression of many neuronal genes including brain-derived neurotrophic factor, Synapsin1 (syn1) and β3-tubulin in BALB/c liver. Furthermore, we show the elevation of the affected proteins in plasma following ASO treatment. Finally, microarray analysis was applied to identify a broad range of genes modulated by REST suppression in mouse liver. Our findings suggest that REST may be an important target for neurodegenerative diseases like Huntington's disease, is also involved in the regulation of a broad range of additional cellular pathways, and that the antisense approach is a viable strategy for selectively modulating REST activity in vivo.
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http://dx.doi.org/10.1089/nat.2013.0445DOI Listing
December 2013

Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs.

Nat Neurosci 2012 Nov 30;15(11):1488-97. Epub 2012 Sep 30.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California, USA.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.
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http://dx.doi.org/10.1038/nn.3230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586380PMC
November 2012

Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis.

Neuron 2012 Jun;74(6):1031-44

Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

The primary cause of Huntington's disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild-type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.
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http://dx.doi.org/10.1016/j.neuron.2012.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383626PMC
June 2012

Genomic analysis of wig-1 pathways.

PLoS One 2012 7;7(2):e29429. Epub 2012 Feb 7.

Department of Drug Discovery, Isis Pharmaceuticals Inc, Carlsbad, California, United States of America.

Background: Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain.

Methods And Results: Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases.

Conclusion: Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029429PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274543PMC
July 2012

Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.

Nat Neurosci 2011 Apr 27;14(4):459-68. Epub 2011 Feb 27.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California, USA.

We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3' untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.
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http://dx.doi.org/10.1038/nn.2779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3094729PMC
April 2011

Heterocyclic replacement of the central phenyl core of diamine-based histamine H3 receptor antagonists.

Eur J Med Chem 2009 Nov 16;44(11):4413-25. Epub 2009 Jun 16.

Johnson & Johnson Pharmaceutical Research and Development, LLC, San Diego, CA 92121, United States.

A series of small molecules consisting of a heterocyclic core flanked by two basic functionalities were synthesized and screened for in vitro affinity at the human histamine H(3) receptor (hH(3)R). Nine of the twenty-eight compounds tested were found to possess a hH(3)R K(i) of less than 5 nM and consisted of a diverse range of central hetero-aromatic linkers (pyridine, pyrazine, oxazole, isoxazole, thiazole, furan, thiophene, and pyrrole). One member of this series, (4-isopropyl-piperazin-1-yl)-(6-piperidin-1-ylmethyl-pyridin-3-yl)-methanone (37), was found to be a high affinity, selective antagonist that crosses the blood-brain barrier and occupies H(3) receptors after oral administration in the rat.
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http://dx.doi.org/10.1016/j.ejmech.2009.06.007DOI Listing
November 2009

Pharmacokinetics and pharmacodynamics of norfluoxetine in rats: Increasing extracellular serotonin level in the frontal cortex.

Pharmacol Biochem Behav 2009 May 5;92(3):469-73. Epub 2009 Feb 5.

Johnson and Johnson Pharmaceutical Research and Development, L. L. C., 3210 Merryfield Row, San Diego, CA 92121, USA.

Norfluoxetine is the most important active metabolite of the widely used antidepressant fluoxetine. Although the pharmacokinetics/pharmacodynamics (PK/PD) relationship and neurochemical profile of fluoxetine is well characterized in human and in animals, little is known about the effect of its metabolite. The aim of this study was to characterize extracellular level of serotonin (5-hydroxytryptamine, 5-HT)-time profile of norfluoxetine after acute administration over 18 h post dose and to establish the relationship between this pharmacodynamic (PD) profile and its pharmacokinetic (PK) properties. Following subcutaneous administration of fluoxetine in rats, plasma and brain PK of fluoxetine and norfluoxetine were monitored respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The extracellular level of 5-HT in the frontal cortex was measured by microdialysis as a PD endpoint. Norfluoxetine when directly administrated to rats caused a significant increase in extracellular level of 5-HT in the frontal cortex and maintained for 18 h. This result is correlated well with higher plasma and brain concentration and longer plasma and brain retention time of norfluoxetine. Our results showed that norfluoxetine contributes to 5-HT transporter inhibition and extends fluoxetine efficacy.
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http://dx.doi.org/10.1016/j.pbb.2009.01.023DOI Listing
May 2009

Novel imidazole-based histamine H3 antagonists.

Bioorg Med Chem Lett 2009 Feb 6;19(3):903-7. Epub 2008 Dec 6.

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

A novel series of imidazole containing histamine H(3) receptor ligands were investigated and found to be potent functional antagonists. After improving the stability of these molecules towards liver microsomes, these compounds were found to have no appreciable affinity for CYP P450s. Subsequent in vivo experiments showed significant brain uptake of (4-chloro-phenyl)-[2-(1-isopropyl-piperidin-4-ylmethoxy)-3-methyl-3H-imidazol-4-yl]-methanone 22.
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http://dx.doi.org/10.1016/j.bmcl.2008.11.114DOI Listing
February 2009

Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81.

J Biol Chem 2009 Jan 1;284(5):2811-2822. Epub 2008 Dec 1.

Johnson & Johnson Pharmaceutical Research & Development, LLC, San Diego, California 92121.

Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.
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http://dx.doi.org/10.1074/jbc.M806409200DOI Listing
January 2009

Dual serotonin transporter inhibitor/histamine H3 antagonists: development of rigidified H3 pharmacophores.

Bioorg Med Chem Lett 2007 Oct 15;17(19):5325-9. Epub 2007 Aug 15.

Johnson & Johnson Pharmaceutical Research and Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

A series of tetrahydroisoquinolines acting as dual serotonin transporter inhibitor/histamine H(3) antagonists is described. The introduction of polar aromatic spacers as part of the histamine H(3) pharmacophore was explored. A convergent synthesis of the final products allowing late stage introduction of the aromatic side chain was developed. In vitro and in vivo data are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2007.08.017DOI Listing
October 2007

Pharmacological characterization of JNJ-28583867, a histamine H(3) receptor antagonist and serotonin reuptake inhibitor.

Eur J Pharmacol 2007 Dec 14;576(1-3):43-54. Epub 2007 Aug 14.

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

Wake-promoting agents such as modafinil are used in the clinic as adjuncts to antidepressant therapy in order to alleviate lethargy. The wake-promoting action of histamine H(3) receptor antagonists has been evidenced in numerous animal studies. They may therefore be a viable strategy for use as an antidepressant therapy in conjunction with selective serotonin reuptake inhibitors. JNJ-28583867 (2-Methyl-4-(4-methylsulfanyl-phenyl)-7-(3-morpholin-4-yl-propoxy)-1,2,3,4-tetrahydro-isoquinoline) is a selective and potent histamine H(3) receptor antagonist (K(i)=10.6 nM) and inhibitor of the serotonin transporter (SERT) (K(i)=3.7 nM), with 30-fold selectivity for SERT over the dopamine and norepinephrine transporters. After subcutaneous administration, JNJ-28583867 occupied both the histamine H(3) receptor and the SERT in rat brain at low doses (<1 mg/kg). JNJ-28583867 blocked imetit-induced drinking (3-10 mg/kg i.p.), confirming in vivo functional activity at the histamine H(3) receptor and also significantly increased cortical extracellular levels of serotonin at doses of 0.3 mg/kg (s.c.) and higher. Smaller increases in cortical extracellular levels of norepinephrine and dopamine were also observed. JNJ-28583867 (3-30 mg/kg p.o.) showed antidepressant-like activity in the mouse tail suspension test. JNJ-28583867 (1-3 mg/kg s.c.) caused a dose-dependent increase in the time spent awake mirrored by a decrease in NREM. Concomitantly, JNJ-28583867 produced a potent suppression of REM sleep from the dose of 1 mg/kg onwards. JNJ-28583867 has good oral bioavailability in the rat (32%), a half-life of 6.9 h and a C(max) of 260 ng/ml after 10 mg/kg p.o. In summary, JNJ-28583867 is a combined histamine H(3) receptor antagonist-SERT inhibitor with in vivo efficacy in biochemical and behavioral models of depression and wakefulness.
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http://dx.doi.org/10.1016/j.ejphar.2007.08.009DOI Listing
December 2007

Pyrrolidino-tetrahydroisoquinolines bearing pendant heterocycles as potent dual H3 antagonist and serotonin transporter inhibitors.

Bioorg Med Chem Lett 2007 Aug 16;17(15):4374-7. Epub 2007 Mar 16.

Johnson & Johnson Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, La Jolla, CA 92121, USA.

A series of novel and potent 6-heteroaryl-pyrrolidino-tetrahydroisoquinolines with dual histamine H(3) antagonist/serotonin transporter inhibitor activity is described. In vitro and in vivo data are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2007.03.043DOI Listing
August 2007

Pyrrolidino-tetrahydroisoquinolines as potent dual H3 antagonist and serotonin transporter inhibitors.

Bioorg Med Chem Lett 2007 May 4;17(9):2603-7. Epub 2007 Feb 4.

Johnson & Johnson Pharmaceutical Research and Development L.L.C., 3210 Merryfield Row, La Jolla, CA 92121, USA.

A series of novel and potent pyrrolidino-tetrahydroisoquinolines with dual histamine H(3) antagonist/serotonin transporter inhibitor activity is described. A highly regio- and diastereoselective synthesis of the pyrrolidino-tetrahydroisoquinoline core involving acid mediated ring-closure of an acetophenone intermediate followed by reduction with NaCNBH(3) was developed. In vitro and in vivo data are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2007.01.106DOI Listing
May 2007

Dual serotonin transporter/histamine H3 ligands: Optimization of the H3 pharmacophore.

Bioorg Med Chem Lett 2007 Feb 2;17(3):702-6. Epub 2006 Nov 2.

Johnson & Johnson Pharmaceutical Research and Development LLC, 3210 Merryfield Row, San Diego, CA 92121, USA.

A series of tetrahydroisoquinolines acting as dual histamine H3/serotonin transporter ligands is described. A highly regio-selective synthesis of the tetrahydroisoquinoline core involving acid mediated ring-closure of an acetophenone intermediate followed by reduction with NaCNBH3 was developed. In vitro and in vivo data are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2006.10.089DOI Listing
February 2007

Identification and pharmacological characterization of prokineticin 2 beta as a selective ligand for prokineticin receptor 1.

Mol Pharmacol 2005 Jun 16;67(6):2070-6. Epub 2005 Mar 16.

Pharmaceutical Research and Development, Johnson & Johnson LLC, 3210 Merryfield Row, San Diego, CA 92121, USA.

Prokineticins 1 and 2 (PK1 and PK2) have been recently identified from humans and other mammals and play multiple functional roles. PK proteins are ligands for two G protein-coupled receptors, PK receptor 1 (PKR1) and PK receptor 2 (PKR2). Here, we report the molecular cloning and pharmacological characterization of an alternatively spliced product of the PK2 gene encoding 21 additional amino acids compared with PK2, designated PK2L (for PK2 long form). PK2L mRNA is broadly expressed, as is PK2. However, PK2L mRNA expression is lower in brain, undetectable in kidney, and much higher in lung and spleen than that of PK2. We expressed PK2L in mammalian cells and characterized the resulting peptide in comparison with PK1 and PK2. Biochemical characterization indicates that secreted PK2L protein is processed into a smaller peptide by proteolytic cleavage. We designate this smaller form of peptide as PK2beta. Coexpression of furin with PK2L significantly increased the PK2beta processing efficiency. Functional studies showed that PK1, PK2, and PK2beta stimulate intracellular Ca(2+) responses in PKR1-expressing cells with similar potencies. However, the PK2beta stimulus of Ca(2+) responses in PKR2-expressing cells is at least 10-fold less potent than that of PK1 or PK2. Differences in receptor selectivity combined with differential tissue expression patterns suggest PK2 and PK2beta might have different functions in vivo. PKRs have been reported to couple to G(q) and G(i) proteins. In this report, we show that PKs not only stimulate Ca(2+) mobilization but also induce cAMP accumulation in PKR-expressing cells.
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http://dx.doi.org/10.1124/mol.105.011619DOI Listing
June 2005

Identification and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid (5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1 (VR1) vanilloid receptor antagonist.

J Med Chem 2005 Mar;48(6):1857-72

Johnson & Johnson Pharmaceutical Research and Development L.L.C., 3210 Merryfield Row, San Diego, California 92121, USA.

High throughput screening using the recombinant human TRPV1 receptor was used to identify a series of pyridinylpiperazine ureas (3) as TRPV1 vanilloid receptor ligands. Exploration of the structure-activity relationships by parallel synthesis identified the essential pharmacophoric elements for antagonism that permitted further optimization via targeted synthesis to provide a potent orally bioavailable and selective TRPV1 modulator 41 active in several in vivo models.
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http://dx.doi.org/10.1021/jm0495071DOI Listing
March 2005

8-OH-DPAT acts on both 5-HT1A and 5-HT7 receptors to induce hypothermia in rodents.

Eur J Pharmacol 2004 Mar;487(1-3):125-32

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Studies using selective drugs and knockout mice have demonstrated that the 5-HT(7) receptor plays an instrumental role in serotonin-induced hypothermia. There is also evidence supporting an involvement of the 5-HT(1A) receptor, although mainly from studies using 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT(1A/7) receptor agonist. Here we studied the effects of 8-OH-DPAT and selective antagonists for the 5-HT(1A) and 5-HT(7) receptors on body temperature in rats, wild-type (5-HT(7)(+/+)) mice and knockout (5-HT(7)(-/-)) mice. At lower doses (0.3-0.6 mg/kg, i.p.), 8-OH-DPAT decreased body temperature in 5-HT(7)(+/+) mice but not in 5-HT(7)(-/-) mice. At a higher dose (1 mg/kg, i.p.) 8-OH-DPAT induced hypothermia in both 5-HT(7)(-/-) and 5-HT(7)(+/+) mice. The 5-HT(1A) receptor antagonist (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide (WAY-100135) (10 mg/kg, i.p.) inhibited the effect of 8-OH-DPAT at all doses in rats and mice. In 5-HT(7)(+/+) mice the selective 5-HT(7) receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol (SB-269970) (10 mg/kg, i.p.) fully inhibited the hypothermia induced by 0.3 mg/kg 8-OH-DPAT, but not that of higher doses. In rats, SB-269970 caused a 60% inhibition of the hypothermia induced by 0.3 mg/kg 8-OH-DPAT. Thus, both 5-HT(7) and 5-HT(1A) receptors are involved in a complex manner in thermoregulation, with the 5-HT(7) receptor being more important at lower, possibly more physiological, concentrations.
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http://dx.doi.org/10.1016/j.ejphar.2004.01.031DOI Listing
March 2004

Characterization of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]acrylamide (JNJ-5207787), a small molecule antagonist of the neuropeptide Y Y2 receptor.

J Pharmacol Exp Ther 2004 Mar 14;308(3):1130-7. Epub 2003 Nov 14.

Johnson & Johnson Pharmaceutical Research and Development, San Diego, CA 92121, USA.

The in vitro pharmacological properties of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]-acrylamide (JNJ-5207787), a novel neuropeptide Y Y(2) receptor (Y(2)) antagonist, were evaluated. JNJ-5207787 inhibited the binding of peptide YY (PYY) to human Y(2) receptor in KAN-Ts cells (pIC(50) = 7.00 +/- 0.10) and to rat Y(2) receptors in rat hippocampus (pIC(50) = 7.10 +/- 0.20). The compound was >100-fold selective versus human Y(1),Y(4), and Y(5) receptors as evaluated by radioligand binding. In vitro receptor autoradiography data in rat brain tissue sections confirmed the selectivity of JNJ-5207787. [(125)I]PYY binding sites sensitive to JNJ-5207787 were found in rat brain regions known to express Y(2) receptor (septum, hypothalamus, hippocampus, substantia nigra, and cerebellum), whereas insensitive binding sites were observed in regions known to express Y(1) receptor (cortex and thalamus). JNJ-5207787 was demonstrated to be an antagonist via inhibition of PYY-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding ([(35)S]GTPgammaS) in KAN-Ts cells (pIC(50) corrected = 7.20 +/- 0.12). This was confirmed auto-radiographically in rat brain sections where PYY-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding was inhibited by JNJ-5207787 (10 microM) in hypothalamus, hippocampus, and substantia nigra. After intraperitoneal administration in rats (30 mg/kg), JNJ-5207787 penetrated into the brain (C(max) = 1351 +/- 153 ng/ml at 30 min) and occupied Y(2) receptor binding sites as revealed by ex vivo receptor autoradiography. Hence, JNJ-5207787 is a potent and selective pharmacological tool available to establish the potential role of central and peripheral Y(2) receptors in vivo.
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http://dx.doi.org/10.1124/jpet.103.060459DOI Listing
March 2004

Non-imidazole heterocyclic histamine H3 receptor antagonists.

Bioorg Med Chem Lett 2003 May;13(10):1767-70

Johnson & Johnson Pharmaceutical Research and Development L. L. C., 3210 Merryfield Row, San Diego, CA 92121, USA.

Continued exploration of the SAR around the lead imidazopyridine histamine H(3) antagonist 1 has led to the discovery of several related series of heterocyclic histamine H(3) antagonists. The synthesis and SAR of indolizine, indole and pyrazolopyridine based compounds are now described.
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http://dx.doi.org/10.1016/s0960-894x(03)00299-3DOI Listing
May 2003

Novel human histamine H(3) receptor antagonists.

Bioorg Med Chem Lett 2002 Nov;12(22):3309-12

Johnson and Johnson Pharmaceutical Research and Development, L.L.C., San Diego, CA 92121, USA.

High throughput screening, using the recombinant human H(3) receptor, was used to identify novel histamine H(3) receptor antagonists. Evaluation of the lead compounds ultimately afforded potent, selective, orally bioavailable compounds (e.g., 38) with favorable blood-brain barrier penetration.
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http://dx.doi.org/10.1016/s0960-894x(02)00738-2DOI Listing
November 2002