Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
  • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention relates to the field of anti-inflammatory agents. More specifically, the present invention provides a novel use for an antisense ohgonucleotide targeted to the coding domain of the acetylcholinesterase (AChE) nucleotide sequence, as an anti-inflammatory agent, particularly for the treatment and/or prevention of inflammation in the joints, central nervous system, gastrointestinal tract, endocardium, pericardium, lung, eyes, skin and urogenital system.
• AChE acetylcholinesterase
• Inflammation plays a crucial role in defense against pathogen invaders as well as in healing and recovery processes following various types of injury.
• the magnitude and duration of inflammatory responses have to be tightly regulated, because excessive inflammatory reactions can be detrimental, leading to autoimmune diseases, neurodegeneration, sepsis, trauma and other pathological conditions.
• regulation of inflammatory reactions is mediated both by immune responses (particularly the secretion of anti-inflammatory cytokines) and by neuroendocrine factors, particularly the activation of the pituitary-adrenal axis and the secretion of glucocorticoids.
• neural mechanisms are also involved in limiting inflammatory responses.
• cholinergic neurons inhibit acute inflammation, providing a rapid, localized, and adaptive anti-inflammatory reflex system (Tracy, 2002).
• ACh acetylcholine
• IL-1 pro-inflammatory cytokines
• IL-6 interleukin-l ⁇
• IL-18 interleukin-l ⁇
• IL-10 anti-inflammatory cytokine IL-10
• IL-1 causes AChE over-production both in PC12 cells and in the rat cortex [Li, Y. et al. (2000) J. Neurosci. 20, 149-155], suggesting a closed loop whereby ACh suppresses IL-1, ablating the induction of AChE production.
• Allostatic breakdown of this intricately controlled pathway may occur under various stressors, including glycinergic (strychnine) or cholinergic agents (succinylcholine), or under myasthenic crisis or post-anesthesia effects [Becker, CM. et al. (1992) Neuron 8, 283-289; Millard, C.B. & Broomfield, CA. (1995) J. Neurochem. 64, 1909- 1918; Subramony, S.H. et al. (1986) Muscle Nerve 9, 64-68; Krasowski, M.D. et al. (1997) Can. J. Anaesth. 44, 525-534].
• glycinergic trychnine
• cholinergic agents succinylcholine
• both injury and chemical stressors induce up-regulation of pro-inflammatory cytokines in the spinal cord (e.g. IL-l ⁇ following experimental spinal injury) or organophosphate inhibitors of acetylcholinesterase (AChE) [Wang, CX. et al. (1997) Brain Res 759, 190-196; Svensson, I. et al. (2001) Neurotoxicology 22, 355-362; Dyer, S.M. et al.
• pro-inflammatory cytokines e.g. IL-l ⁇ following experimental spinal injury
• AChE organophosphate inhibitors of acetylcholinesterase
• a parallel stress response involves down-regulation of choline acetyltransferase (ChAT) [Kaufer, D. et al. (1998) Nature 393, 373-377] and the genomically linked vesicular acetylcholine transporter (VAChT) [Weihe, E. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3547-3552], together limiting the production and vesicle packaging of acetylcholine while expediting its degradation. This yields down-regulation of the cholinergic hyperexcitation that is associated with many stresses.
• ChAT choline acetyltransferase
• VAChT genomically linked vesicular acetylcholine transporter
• this stress response is associated with hypersensitivity to both agonists and antagonists of cholinergic neurotransmission [Meshorer, E. et al. (2002) Science 295, 508-512] and abnormal locomotor activities that can be ablated under antisense destruction of AChE-R mRNA [Cohen, O. et al. (2002) Mol. Psychiatry 7, 874-885]. Finely-tuned control over AChE-R levels thus emerged as a key component of stress management by spinal cord motoneurons. AChE-R over-expression, which suppresses ACh levels, further lead to increased IL-1 production. Should this be the case, antisense suppression of AChE-R production [Brenner, T. et al. (2003) Faseb J. 17(2), 214-22] would increase ACh levels and reduce the levels of pro-inflammatory cytokines in CNS neurons.
• Endotoxin administration induces fever, malaise and increased production and secretion of cytokines, particularly TNF- ⁇ , IL-6, IL-1 and IL-lra and cortisol [for review see Burrell R. (1994) Circ. Shock 43:137-53], as well as proteases [Fahmi H. and Chaby R. (1994) Immunol. Invest. 23:243-58].
• cytokines particularly TNF- ⁇ , IL-6, IL-1 and IL-lra and cortisol
• proteases Flahmi H. and Chaby R. (1994) Immunol. Invest. 23:243-58.
• endotoxin-induced cytokine secretion is correlated with impairments in verbal and non-verbal declarative memory functions [Reichenberg A. et al. (2001) Arch. Gen. Psychiatry 58:445-52].
• cholinergic processes are relevant to endotoxin responses because in the central nervous system (CNS), cholinergic responses are notably involved in several important aspects of cognitive functioning, including attention, learning and memory (for reviews see Levin E. D. and Simon B. B. (1998) Psychopharmacology (Berl) 138:217-30; Segal M. and Auerbach J. M. (1997) Life Sci. 60:1085-91].
• endotoxin decreases brain choline acetyltransferase activity [Willard L. B. et al. (1999) Neuroscience 88:193-200], similar to the effects of psychological stress [Kaufer (1998) id ibid.].
• endogenous or exogenous acetylcholine attenuates the release of pro-inflammatory cytokines from endotoxin- stimulated human macrophages [Borovikova (2000) id ibid.; Bernik (2002) id ibid.; Tracey (2001) id ibid.].
• the ACh hydrolyzing enzyme acetylcholinesterase was considered as potentially being of particular relevance to these processes because AChE controls ACh levels and since AChE inhibitors improve cognitive functions in both clinical and experimental paradigms [Palmer A. M. (2002) Trends Pharmacol. Sci. 23:426-33; Weinstock M. (1995) Neurodegeneration 4:349-56].
• AChE over-expression is triggered by acute and chronic stressful insults [Meshorer (2002) id ibid.] and induces progressive memory impairments, as was demonstrated in transgenic mice [Beeri R. et al. (1995) Curr. Biol. 5:1063-71].
• AChE-R Stress-induced transcriptional activation of AChE gene expression is associated with accumulation of the normally rare "readthrough" AChE-R splice variant [Soreq and Seidman (2001) id ibid.]. In the short range, the AChE-R excess reduces the stress-induced cholinergic hyperexcitation [Kaufer (1998) id ibid.]; in the long range, it induces hypersensitivity to cholinergic agonists and antagonists [Meshorer (2002) id ibid.]. Mice that overexpress both AChE-S and AChE-R present progressive dendritic and spine loss [Beeri R. et al. (1997) J. Neurochem.
• mice display early-onset deficits in social recognition and exaggerated responsiveness to stressful insults. These can be briefly ameliorated by conventional anticholinesterase treatment or for longer periods by an antisense ohgonucleotide capable of specifically inducing the destruction of AChE-R mRNA [Cohen (2002) id ibid.], suggesting that AChE-R is the primary cause. Thus, AChE-R production may lead to both positive and negative effects on cognition.
• the present inventors have previously found that antisense oligonucleotides against the common coding region of AChE are useful for suppressing AChE-R production [see WO 98/26062].
• the inventors have shown the use of an antisense ohgonucleotide against the AChE sequence for the treatment of myasthenia gravis [WO 03/002739 and US 10/402,016].
• the present invention provides a novel use for an antisense ohgonucleotide directed against the AChE mRNA sequence, as a new anti-inflammatory agent.
• the present invention refers to the use of an inhibitor of AChE expression, as an anti-inflammatory agent.
• said inhibitor of AChE expression is an antisense ohgonucleotide directed against AChE, having any one of the following sequences: 5' CTGCCACGTTCTCCTGCACC 3' (SEQ. ID. NO:l);
• the invention provides the use of an inhibitor of AChE as defined herein, as a suppressor of pro-inflammatory cytokines release.
• said inhibitor of AChE is the antisense ohgonucleotide denoted by SEQ. ID. NO. 1.
• the present invention intends to provide a pharmaceutical composition for the treatment of conditions triggering an inflammatory response, comprising as active agent the above-defined inhibitor of AChE expression.
• the composition further comprises additives, carriers and/or diluents.
• said inhibitor of AChE expression is an antisense ohgonucleotide directed against AChE. More preferably, said antisense nucleotide has the sequence as denoted in SEQ. ID. NO:l.
• the present invention provides a pharmaceutical composition for the treatment and/or prevention of inflammation in the joints, central nervous system, gastrointestinal tract, endocardium, pericardium, lung, eyes, skin and urogenital system, comprising as active agent the inhibitor of AChE expression as defined above, optionally further comprising any one of additives, carriers and/or diluents.
• said inhibitor of AChE expression is an antisense ohgonucleotide. More preferably, said ohgonucleotide has the sequence as denoted in SEQ. ID. NO:l.
• the inhibitor of AChE expression is to be used in the preparation of the pharmaceutical composition of the invention.
• the invention teaches a method of treatment of conditions triggering an inflammatory response, wherein said method comprises administering an effective amount of an inhibitor of AChE expression, as defined herein, or a composition comprising as active agent an inhibitor of AChE expression, prepared as described in the description.
• said inhibitor of AChE expression to be used in the method of the invention is an antisense ohgonucleotide, which, more preferably, has the sequence denoted by SEQ. ID. NO:l.
• Figure 1A-F Reduced VAChT accumulation in cholinergic terminals and partition cells of treated monkeys.
• Fig. IB Average value of volume and average number per cell of labeled terminals, including all motoneurons detected in a section.
• Fig. IC Population distribution of volume and average number per cell of labeled terminals, including all motoneurons detected in a section.
• Fig. ID Average values of Figs. IB, IC analyses (+ Standard Evaluation of the
• Fig. IE Immunolabeling with anti-ChAT antibody in partition cells from na ⁇ ve spinal cord, localized in close proximity to the central canal (arrows).
• Hematoxylin was used for background staining.
• Fig. IF Higher magnification of ChAT positive partition cells in na ⁇ ve monkeys (1) or following oral (p.o.) administration of 150 ⁇ g/Kg/day (2) or 500 ⁇ g/Kg/day (3) and i.v. administration of 500 ⁇ g/Kg/day hENlOl (4). Note dose- independent handling - induced reductions in both terminals volume and density.
• n. na ⁇ ve
• Term. terminal
• vol. volume
• Part. Ce. Partition cell
• Cent. Can. Central canal.
• Figure 2A-J Selective AChE-R mRNA suppression by hENlOl in monkey spinal cord neurons.
• Fig. 2A Scheme of the human ACHE gene coding exons and two of its alternative transcripts, the synaptic AChE-S (S) and the stress -associated
• AChE-R (R) mRNA The S transcript includes exons 2, 3, 4 and 6, whereas the
• R transcript contains exons 2, 3, 4, 5 and pseudointron 4'. These distinctions served to prepare transcript-specific probes, indicated by an asterisk.
• Fig. 2B Sampling site on the dissected monkey lumbar spinal cord is indicated by an arrow.
• Fig. 2C- J Tissue sections from lumbar spinal cords were prepared following 7- day treatment with the noted doses of hENlOl by p.o. or i.v. administration.
• Fig. 2C No treatment, staining specific for AChE-R mRNA.
• Fig. 2D No treatment, staining specific for AChE-S mRNA.
• Fig. 2E Treatment with 150 ⁇ g/kg/day of EN101, p.o., staining specific for
• Fig. 2F Treatment with 150 ⁇ g/kg/day of EN101, p.o., staining specific for
• FIG. 2G Treatment with 500 ⁇ g/kg/day of ENIOI, p.o., staining specific for
• Fig. 2H Treatment with 500 ⁇ g/kg/day of ENIOI, p.o., staining specific for
• Fig. 21 Treatment with 500 ⁇ g/kg/day of ENIOI, i.v., staining specific for
• Fig. 2J Treatment with 500 ⁇ g/kg/day of ENIOI, i.v., staining specific for
• Figure 3A-C Cell size-dependent efficacy of neuronal AChE-R mRNA suppression.
• Fig. 3A Scheme of the lumbar spinal cord and its three compartments: the ventral and dorsal horns separated by the intermediate zone and the central canal.
• Fig. 3C Shown are fractions of AChE-R positive neurons from the three size groups under the different treatment regimens. Insets: representative neurons from the different size groups, taken from the p.o. 150 ⁇ g/Kg/day regimen. Columns show average AChE-R positive cells in each size group + SEM representing repeated analyses of the entire lumbar spinal cord gray matter in multiple sections. Stars note significant differences (p ⁇ 0.05, Wilcoxon test). Abbreviations: Cent. Can., central canal; D. h., dorsal horn; I. z., Intermediate zone; V. h., ventral horn; pos. ce., positive cells; si. gr., size group; Ce. Bo. Diam., cell body diameter.
• Figure 4A-C Suppression of stress-induced neuronal pro- inflammatory cytokines under antisense intervention with AChE-R expression.
• Fig. 4C Fractions of IL-6 positive spinal cord neurons were evaluated essentially as under 4A. Note decreases in both IL-l ⁇ and IL-6 in spinal cord neurons of monkeys treated with 500 ⁇ g/Kg/day EN101. Abbreviations: pos. ce., positive cells.
• Figure 5A-D Changes over time in the human plasma levels of AChE activity and in AChE-R cleavage.
• Fig. 5A Hydrolytic activities. Shown are plasma AChE activities (mean + SEM) for ten volunteers injected twice, with endotoxin or saline (placebo) at the noted intervals after injection. Pre-injection (baseline) AChE level was considered as 100% for each individual. Asterisks denote statistical difference (p ⁇ 0.05).
• Fig. 5B Immunoblot. Shown are consecutive results for one individual. Plasma samples underwent electrophoresis by SDS-PAGE, and the blot immunoreacted with anti-AChE-R antibodies. Note the 6.5kDa AChE-R cleavage product. Left lanes indicate the response to a placebo injection; right lanes demonstrate elevated AChE-R cleavage in response to endotoxin.
• Fig. 5C Densitometric intensities. Shown are average values (mean + SEM) of the rapidly migrating AChE-R cleavage product in plasma of the endotoxin and placebo treated individuals as % of baseline (described in A). Note: Elevated AChE-R cleavage in endotoxin-treated subjects co-appeared with decreased AChE activity.
• Figure 7A-C AChE-R is expressed in human vascular endothelial cells from various tissues.
• Fig. 7A AChE-R mRNA. Shown are the results of in situ hybridization using a 5'-biotinylated cRNA probe selective for the AChE-R mRNA variant on sections of human vascular endothelial cells affected by an inflammatory process (skin hypersensitivity vasculitis; labeling is seen as pink color, red arrow).
• Fig. 7B AChE-R protein. Shown is an immunomicrograph of human kidney vascular endothelial cells from a patient with vascuhtis, labeled with antibodies targeted at the AChE-R C-terminal peptide (red arrow).
• Fig. 7C Image analvsis.
• AChE-R mRNA and AChE-R protein labeling intensities black and white columns, respectively
• k. rej. kidney rejection
• k. vas. kidney vascuhtis
• nonspec, non-specific n. end., normal endothelium
• m., muscle hyp. vase, hypersensitivity vascuhtis.
• Figure 8A-C Bidirectional associations between AChE-R cleavage and the changes in cortisol and cytokines.
• Fig. 8A cortisol.
• Fig. 8B TNF- ⁇ .
• Fig. 8C IL-6.
• r correlation coefficient
• t time after injection
• Plac placebo
• end. endotoxin
• H. p. inj. hours post-injection
• cleav. prod. cleavage product.
• Figure 10 Endotoxin-induced improvement in working memory.
• r correlation coefficient
• t time after injection
• S.b. Span backward
• plac placebo
• endot. endotoxin
• H.p.inj. hours post-injection
• cleav. prod. cleavage product
• act. activity.
• Figure 11A-C Scheme - Endotoxin induces interrelated cytokine- cholinergic effects on memory.
• Fig. IIA At 1 hr post-treatment: Endotoxin induces the release of cytokines, cortisol and proteases. Cytokines elevation associates with impaired declarative memory, which is a medial temporal lobe - associated phenomenon. Cortisol induces AChE-R production, which elevates the immunopositive AChE-R amounts in plasma.
• Vesicular ACh is released into the synaptic cleft, where it affects neuronal electrophysiology and may improve working memory, which is a neocortex - associated property.
• ACh begins to suppress cytokines production in macrophages (circular arrow).
• Fig. IIB At 3 hr post-treatment: Proteases release a C-terminal fragment of 36 amino acids in length from AChE-R and initiate further destruction, followed by decreases in AChE activity. Endotoxin is already gone, and ACh effectively suppresses cytokines production; Increased ACh levels (reflecting enhanced secretion and the decrease in AChE's hydrolytic activity) are probably associated with activated working memory, whereas the elevation in
• AChE-R cleavage product is associated with a lower working memory improvement.
• Fig. 11C At 9 hr post-treatment: Cortisol is gone as well. However, the persistent, although slow decrease in AChE activity is associated both with the impaired declarative memory and, probably through ACh increases, with the activated working memory. The steady increase in AChE-R cleavage product is now associated both with a greater impairment in declarative memory and with lower improvement in working memory.
• FIG. 12A-B Transgenic mice display higher body temperature than wild-type mice.
• Fig. 12A Graph showing the temperature of each mouse over time, squares represent transgenic mice, circles, control.
• Fig. 12B Graph showing the average temperature of each group (transgenic or control) over time, diamonds represent transgenic mice, squares, control.
• Figure 13A-C Effects of Tacrine on LPS-induced IL-1 secretion in the hippocampus and IL-1 and TNF- ⁇ secretion in the serum.
• Fig. 13A Graph showing the levels of IL-l ⁇ in the hippocampus.
• Fig. 13B Graph showing the levels of IL-l ⁇ in the serum.
• Fig. 13C Graph showing the levels of TNF- ⁇ in the serum.
• Figure 14A-C Effects of Rivastigmine on LPS-induced IL-1 secretion in the hippocampus and IL-1 and TNF- ⁇ secretion in the serum.
• Fig. 14A Graph showing the levels of IL-l ⁇ in the hippocampus.
• Fig. 14B Graph showing the levels of IL-l ⁇ in the serum.
• Fig. 14C Graph showing the levels of TNF- ⁇ in the serum.
• Figure 15A-H Effects of surgery stress on emotional and cognitive parameters.
• Fig. 15A Graph showing the effect of surgery stress on anxiety.
• Fig. 15B Graph showing the effect of surgery stress on depression.
• Fig. 15C Graph showing the effect of surgery stress on fatigue.
• Fig. 15D Graph showing the effect of surgery stress on pain.
• Fig. 15E Graph showing the effect of surgery stress on word list recall.
• Fig. 15F Graph showing the effect of surgery stress on word list recognition.
• Fig. 15G Graph showing the effect of surgery stress on story recall.
• Fig. 15H Graph showing the effect of surgery stress on figure recall.
• Figure 16A-C Effect of surgery stress on cytokine levels.
• Fig. 16A Graph showing the effect of surgery stress on IL-1 and IL-6 levels.
• Fig. 16B Correlation between IL-1 and depression.
• Fig. 16C Correlation between cytokines and cognitive parameters.
• Figure 17A-C Reduction of AChE gene expression upon EN301 treatment.
• Fig. 17A Analysis of RT-PCR reaction (AChE exon 2 product after 31 PCR cycles). From left to right: lane 1, marker; lanes 2-8, samples from EN301- treated mice; lanes 9-14, samples from PBS-treated mice.
• Fig. 17B Histogram representing quantitative analysis of the results obtained in the PCR reaction using primers targeting the common sequence in exon 2 of murine AChE cDNA.
• Fig. 17C Histogram representing quantitative analysis of the results obtained in the PCR reaction using primers targeting the sequence in exon 6 unique to the AChE-S variant.
• - AChE-R acetylcholinesterase, "readthrough” variant or isoform, its mRNA includes pseudo-intron 14
• AChE-S acetylcholinesterase, synaptic variant or isoform
• CNS central nervous system EN101: may also be referred as AS3, antisense ohgonucleotide targeted against human, rat or mouse (hENlOl, rENlOl or mENlOl, respectively)
• AS3 antisense ohgonucleotide targeted against human, rat or mouse (hENlOl, rENlOl or mENlOl, respectively)
• - EN301 may also be referred as mENlOl, antisense ohgonucleotide targeted against mouse AChE mRNA i.v.: intravenous i.p.: intraperitoneal
• Antisense ohgonucleotide A nucleotide comprising essentially a reverse complementary sequence to a sequence of AChE mRNA.
• the nucleotide is preferably an oligodeoxynucleotide, but also ribonucleotides or nucleotide analogues, or mixtures thereof, are contemplated by the invention.
• the antisense ohgonucleotide may be modified in order to enhance the nuclease resistance thereof, to improve its membrane crossing capability, or both.
• the antisense ohgonucleotide may be linear, or may comprise a secondary structure. It may also comprise enzymatic activity, such as ribozyme activity.
• hENlOl SEQ. ID. NO:l
• hENlOl a 2'- oxymethylated antisense ohgonucleotide inducing AChE-R mRNA destruction.
• hENlOl prevented the stress-induced increases in plasma AChE activities and selectively suppressed neuronal AChE-R mRNA and interleukins -l ⁇ and -6 levels in a dose- and cell size-dependent manner.
• the present invention refers to the use of an inhibitor of AChE expression, as an anti-inflammatory agent.
• said inhibitor of AChE expression is any agent which is capable of blocking or hindering the expression of the AChE gene, particularly by interacting with its mRNA.
• said inhibitor may be an AChE-specific ribozyme, a double -stranded nucleotide sequence used for RNA interference of the AChE gene, or an antisense ohgonucleotide directed against AChE.
• Antisense nucleotides are preferably nuclease resistant.
• said inhibitor of AChE expression selectively inhibits the AChE-R mRNA, consequently selectively inhibiting the expression of the AChE-R isoform.
• any agent capable of inhibiting the soluble AChE-R isoform may also be an anti-inflammatory agent. Therefore, a putative molecule that could block AChE-R expression and/or function would be an anti- inflammatory agent.
• AChE-S mRNA appeared in processes of many more spinal cord neurons than AChE-R mRNA, creating a pattern reminiscent of VAChT labeling in the rat spinal cord ventral horn [Weihe et al. (1996) id ibid.].
• hENlOl treatment was highly efficient with neuronal AChE-R mRNA and much less effective with AChE-S mRNA.
• the reduced intensity of neuronal AChE-S mRNA labeling likely reflected limited reduction in neuronal AChE-S mRNA levels as well.
• AChE-S mRNA in processes was reduced, suggesting common tendency for reduced dendrite translocation of the rodent and primate AChE-S mRNA transcript under stress [Meshorer et al. (2002) id ibid.]. This difference further strengthened the notion that the na ⁇ ve monkey was indeed under no stress, an important fact in a study with strictly limited number of animals.
• the reduced AChE-S mRNA in neuronal processes of the treated monkeys may be treatment- and/or drug-induced. Following 7 days treatment, a shift from the primary AChE-S mRNA transcript to the stress- induced antisense-suppressible AChE-R mRNA may be visualized in the neuronal processes (Fig. 2A-2J).
• said inhibitor of AChE expression is an antisense ohgonucleotide directed against AChE, having any one of the following sequences:
• antisense oligonucleotides denoted by SEQ. ID. NO:l or SEQ. ID. NO:7 are also referred to herein as EN101, or hENlOl.
• the antisense oligonucleotides directed against AChE have been described in the past by the present inventors [WO 03/002739], and were shown to have a potent effect in the treatment of the neuromuscular pathology myasthenia gravis [applicant's co-pending US 10/402,016].
• the antisense ohgonucleotide directed against AChE was able to reduce the release of IL-l ⁇ , which is a pro-inflammatory cytokine.
• Example 1 AChE-R mRNA levels in motoneurons were minimally affected, However, ehmination of AChE-R production in spinal cord smaller neurons potentially increased ACh signaling within the treated tissue, in spite of. the stress-induced reduction in VAChT and ChAT [Kaufer et al. (1998) id ibid.]. This attributes to AChE-R the primary role of regulating ACh levels in the CNS. Findings of others show large variability in the electrophysiological activity patterns of spinal cord interneurons [Perlmutter (1996) id ibid.] as well as pre-movement instructed delay activity in them [Prut and Fetz (1999) id ibid.].
• AChE-R expression in small cholinergic neurons may thus contribute to the control of motoneuron activities (e.g. motor reflexes).
• C-terminal structures, which affect the chohnergic input to motoneurons, were considered to originate in proximity to the motoneurons themselves [Hellstrom (1999) id ibid.]. This study attributes this origin to AChE mRNA positive interneurons and small cholinergic neurons located in the ventral horn and intermediate zone of the lumbar spinal cord.
• antisense oligonucleotides directed against AChE have also been described, and potentially have the same anti-inflammatory effect as hENlOl, as demonstrated in Example 16 for mENlOl.
• antisense oligonucleotides derived from the mouse and the rat AChE homologous sequences which have the following sequences:
• mENlOl 5'-CTGCAATATTTTCTTGCACC-3' SEQ. ID. NO:2
• EN301 also referred herein as EN301.
• Example 16 demonstrates how administration of mENlOl (EN301) was able to reduce the levels of AChE-R in the brain. This could be done directly, upon crossing the blood-brain-barrier, or indirectly, by reducing the levels of peripheral AChE, increasing the levels of ACh, which would then suppress the production of pro-inflammatory cytokines by macrophages.
• the invention provides the use of an inhibitor of AChE as defined herein, as a suppressor of pro-inflammatory cytokines release.
• said inhibitor of AChE is the antisense ohgonucleotide denoted by any one of SEQ. ID. NO:l, SEQ. ID. NO:2 and SEQ. ID. NO:7.
• said inhibitor of AChE is the antisense ohgonucleotide denoted by SEQ. ID. NO:l or SEQ. ID. NO:7.
• IL-l ⁇ is the pro-inflammatory cytokine to be suppressed by the antisense ohgonucleotide denoted by any one of SEQ. ID. NO:l, SEQ. ID. NO:2 and SEQ. ID. NO:7.
• Pro-inflammatory cytokine release may be triggered by factors of acquired, chemical or genetic origin. Amongst others, these may be stress, bacterial infection, drugs, irradiation, exposure to AChE inhibitors, stroke, auto-immune diseases, multiple chemical sensitivity, or any cumulative age-dependent damages.
• Known conditions which trigger pro-inflammatory cytokine release are bacterial infection, drugs, irradiation, exposure to AChE inhibitors, stroke, auto-immune diseases, multiple chemical sensitivity, or any cumulative age- dependent damages.
• Stress-induced spinal IL-l ⁇ over-production and spinal IL-l ⁇ suppression following AS-ON inhibition of AChE-R support the notion of cholinergic regulation of anti-inflammatory response in the CNS.
• "stressed" neurons produce high levels of AChE-R, reducing ACh and allowing uninterrupted production of IL-l ⁇ in CNS neurons that do not express IL-l ⁇ under normal conditions.
• Antisense suppression of the stress-induced AChE-R would increase ACh levels, which can then suppress IL-l ⁇ production in CNS neurons.
• Such cholinergic regulation of inflammatory response within the CNS may explain both the increase of pro-inflammatory cytokines under cholinergic imbalance (e.g.
• the invention also provides the use of an inhibitor of AChE expression, as defined herein, as an inducer of cartilage regeneration.
• the antisense oligodeoxynucleotides used as anti-inflammatory agents in the present invention are preferably nuclease resistant. There are a number of modifications that impart nuclease resistance to a given ohgonucleotide.
• oligonucleotides may be made nuclease resistant e.g., by replacing phosphodiester internucleotide bonds with phosphorothioate bonds, replacing the 2'-hydroxy group of one or more nucleotides by 2'-O-methyl groups, or adding a nucleotide sequence capable of forming a loop structure under physiological conditions to the 3' end of the antisense ohgonucleotide sequence.
• An example for a loop forming structure is the sequence 5' CGCGAAGCG, which may be added to the 3' end of a given antisense ohgonucleotide to impart nuclease resistance thereon.
• Phosphorothioate-modifled oligonucleotides are generally regarded as safe and free of side effects.
• the antisense oligonucleotides of the present invention have been found to be effective as partially phosphorothioates and yet more effective as partially 2'-O-methyl protected oligonucleotides.
• WO 98/26062 teaches that AChE antisense oligonucleotides containing three phosphorothioate bonds out of about twenty internucleotide bonds are generally safe to use in concentrations of between about 1 and 10 ⁇ M. However, for long-term applications, oligonucleotides that do not release toxic groups when degraded may be preferred.
• inhibitor of AChE as defined above may also be used as an antipyretic.
• said inhibitor of AChE is the antisense ohgonucleotide denoted by any one of SEQ. ID. NO:l, SEQ. ID. NO:2 and SEQ. ID. NO:7.
• transgenic mice with host AChE-R elevation show inherently higher body temperature as compared to strain, gender and age-matched controls. Furthermore, their body temperature remains higher also under anesthesia, demonstrating impaired regulation and tentative association of AChE-R with pyrogenic responses. Thus, inhibitors of AChE-R expression would also have an effect in lowering the elevated body temperature that is characteristic of inflammatory reactions.
• the dosage of the antisense oligodeoxynucleotide is about 0.001 to 50 ⁇ g ohgonucleotide per gram of body weight of the treated mammalian subject.
• the dosage is about 0.01 to about 5.0 ⁇ g/g. More preferably, the dosage is between about 0.05 to about 0.7 ⁇ g/g.
• the optimal dose range is between 50-500 ⁇ g/kg of body weight of the treated subject, for rats, monkeys and also humans.
• the present invention intends to provide a pharmaceutical composition for the treatment of conditions triggering an inflammatory response in a mammalian subject in need, comprising as active agent the above-defined inhibitor of AChE expression.
• the composition further comprises pharmaceutically acceptable additives, carriers and/or diluents.
• said inhibitor of AChE expression is an antisense ohgonucleotide directed against AChE.
• said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:l and SEQ. ID. NO:7.
• said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:2 and SEQ. ID. NO:3.
• the present invention provides a pharmaceutical composition for the treatment and/or prevention of inflammation in the joints, central nervous system, gastrointestinal tract, endocardium, pericardium, lung, eyes, skin and urogenital system in a mammalian subject in need, comprising as active agent the inhibitor of AChE expression as defined above, optionally further comprising pharmaceutically acceptable additives, carriers and/or diluents.
• said inhibitor of AChE expression is an antisense ohgonucleotide. More preferably, wherein said mammalian subject is a human, said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:l and SEQ. ID. NO:7.
• said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:2 and SEQ. ID. NO:3.
• the inhibitor of AChE expression as defined above, is to be used in the preparation of the pharmaceutical composition of the invention.
• the antisense ohgonucleotide of the invention is generally provided in the form of pharmaceutical compositions.
• Said compositions are for use by injection, topical administration, or oral uptake.
• the pharmaceutical composition of the invention may comprise as active agent a combination of at least two antisense oligonucleotides as defined in the invention, or functional analogs, derivatives or fragments thereof.
• analogs and derivatives is meant the “fragments”, “variants”, “analogs” or “derivatives” of said nucleic acid molecule.
• a “fragment” of a molecule such as any of the ohgonucleotide sequences of the present invention, is meant to refer to any nucleotide subset of the molecule.
• a “variant” of such molecule is meant to refer a naturally occurring molecule substantially similar to either the entire molecule or a fragment thereof.
• An “analog” of a molecule can be without limitation a paralogous or orthologous molecule, e.g. a homologous molecule from the same species or from different species, respectively.
• Preferred uses of the pharmaceutical compositions of the invention by injection are subcutaneous injection, intraperitoneal injection, intravenous and intramuscular injection.
• the pharmaceutical composition of the invention generally comprises a buffering agent, an agent which adjusts the osmolarity thereof, and optionally, one or more carriers, excipients and/or additives as known in the art, e.g., for the purposes of adding flavors, colors, lubrication, or the like to the pharmaceutical composition.
• a preferred buffering agent is Tris, consisting of 10 mM Tris, pH 7.5-8.0, which solution is also adjusted for osmolarity.
• the antisenses are suspended is sterile distilled water or in sterile saline.
• Carriers may include starch and derivatives thereof, cellulose and derivatives thereof, e.g., microcrystalline cellulose, xantham gum, and the like.
• Lubricants may include hydrogenated castor oil and the like.
• compositions for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
• Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
• compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
• compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
• compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self- emulsifying semisolids.
• compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Such compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
• the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
• the pharmaceutical compositions may be formulated and used as foams.
• Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
• the pharmaceutical composition of the invention is for daily use by a subject in need of such treatment, at a dosage of active ingredient between about O.OOl ⁇ g/g and about 50 ⁇ g/g.
• the treatment and/or prevention comprises administering a dosage of active ingredient of about 0.01 to about 5.0 ⁇ g/g.
• said dosage of active ingredient is of between about 0.05 to about 0.70 ⁇ g/g, and even most preferably , the dosage is from 0.15 to 0.50 ⁇ g/g of body weight of the subject in need.
• the antisense agent targeted toward the human ACHE sequence appeared effective in Cynomolgus monkeys at the same nanomolar dose as that of the corresponding agents in mice [Cohen et al. (2002) id ibid.] and rats Brenner et al. (2003) id ibid.].
• Long-term AChE-R overproduction is associated with impaired locomotion control that is susceptible to improvement under antisense suppression of AChE-R production [Shohami (2000) id ibid.].
• the invention teaches a method of treatment of conditions triggering an inflammatory response, wherein said method comprises administering a therapeutically effective amount of an inhibitor of AChE expression to a mammalian subject in need, as defined herein, or a composition comprising as active agent an inhibitor of AChE expression, prepared as described above.
• said inhibitor of AChE expression to be used in the method of the invention is an antisense ohgonucleotide, which, more preferably wherein said mammalian subject is a human, said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:l and SEQ. ID. NO:7.
• said antisense nucleotide has the sequence as denoted by any one of SEQ. ID. NO:2 and SEQ. ID. NO:3.
• Said therapeutic effective amount, or dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
• Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50, found to be effective in in vitro as well as in in vivo.
• the variant specificity, low dose and long duration efficacy of the antisense agents may be clear advantages over conservative drugs, both for interfering with acute stress-induced symptoms and inflammatory response, and hence for prevention of neurodeterioration.
• These considerations may be relevant to various disease conditions, including amyotrophic lateral sclerosis [Shaw, P.J. & Eggett, C.J. (2000) J. Neurol. 247 Suppl 1: 117-27], myasthenic syndromes [Becker et al. (1992) id ibid.], muscular dystrophy [Cifuentes-Diaz, C. et al. (2001) J. Cell Biol. 152: 1107-1114], spinal muscular atrophy [Sendtner, M.
• Figure 11 presents a scheme summarizing the kinetic follow-up for the different parameters that were measured and the postulated associations between them, predicting potentially causal relationships between the induction of cytokines, hormone secretion, AChE modulations and the resultant memory changes.
• the endotoxin-induced impairment in declarative memory was highest and correlated positively with cytokine secretion, whereas the improvement in working memory became prominent at 3hr post-treatment and showed no correlation with cytokine secretion.
• both types of memory changes were significantly correlated with AChE-R cleavage, although cholinergic control over working memory seemed to begin earlier than for declarative memory (3 hr vs. 9 hr post-injection, Fig. IIB and Fig. 11C, respectively).
• Test substance Human (h) HPLC-purified, GLP grade EN101 (purity 95% as verified by capillary electrophoresis) was purchased from Avecia Biotechnology (Milford, MA). The primary hENlOl sequence,
• 5'CTGCCACGTTCTCCTGCA * C * C * 3' is complementary to the coding sequence of human AChE mRNA (GeneBank Accession No. NM 000665, nucleotide positions 733-752) within exon 2, common to all three AChE variants [Soreq, H. & Zakut, H. (1993) Human cholinesterases and anticholinesterases, Academic Press, INC. San Diego; Ben Aziz-Aloya, R. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2471-2475].
• the three 3'-terminal residues ( * ) were protected against nuclease attack with oxymethyl groups at the 2' position.
• the sequence representing hENlOl with the three 3'-terminal bases modified is denoted by SEQ. ID. NO:7. Lyophilized oligonucleotides were resuspended in sterile double distilled water (24 mg/ml), and stored at -20 °C.
• hENlOl stability Stability of freeze-dried hENlOl was tested by HPLC during storage at -20 ⁇ 5°C, 4 ⁇ °C and 25 ⁇ 2°C (60 ⁇ 5% relative humidity) in the dark. Three samples from each storage condition were collected after 3, 6 and 9 months and their stability analyzed by HPLC hENlOl was found to be stable for at least 6 months at -20°C under these storage conditions.
• hENlOl administration Three pairs of 1.5 to 2.5 Kg cynomolgus monkeys, 1 male and 1 female, were administered hENlOl for 7 days: 150 ⁇ g/Kg daily per os (p.o.) by oral gavage (15 ⁇ g/ml in 0.9% saline) or 500 ⁇ g/Kg daily (p.o., 50 ⁇ g/ml in saline) or by intravenous (i.v.) injection (100 ⁇ g/ml in saline). Plasma samples were removed at the noted hours following the second day of treatment and kept at -20°C until use. Following 1 week of daily treatment, animals were euthanized and lumbar spinal cord preparations were paraffin- embedded by standard procedures. One male na ⁇ ve monkey served as control.
• Toxicology Potential toxicity of hENlOl was tested at Huntingdon before, during and following treatment. Among the parameters noted were body weight, food consumption, general locomotor behavior, electrocardiography and blood pressure, blood count, prothrombin time and standard blood chemistry (Hitachi 917 Clinical Chemistry Analyzer). Post mortem observation included organ weights and scanning of hematoxylin and eosin-stained sections of brain, heart, kidneys, liver, lungs, spinal cord and stomach.
• Hybridization was performed overnight at 52°C in hybridization mixture containing 10 ⁇ g/ml probe, 50 ⁇ g/ml yeast tRNA, 50 ⁇ g/ml heparin and 50% formamide in 375 mM Na chloride, 37.5 mM Na citrate, pH 4.5. Slides were washed to remove unhybridized probe, blocked with 1% skim milk containing 0.01% Tween-20 and 2 mM levamisol, an alkaline phosphatase inhibitor used to suppress non-specific staining and incubated with streptavidin-alkaline phosphatase (Amersham Pharmacia, Little Chalfont Bucks, UK). Fast RedTM substrate (Roche Diagnostics, Mannheim, Germany) was used for detection.
• Immunohistochemistry Re-hydrated spinal cord sections were subjected to heat-induced antigen retrieval by microwave treatment in 0.01 M citrate buffer, pH 6.0. Non-specific binding was blocked by 4% naive goat or donkey serum in PBS with 0.3% Triton X-100 and 0.05% Tween 20. Slides were incubated with primary antibodies diluted in the same buffer (1 h, room temp., overnight, 4°C). Sections were rinsed and incubated with biotin-conjugated secondary antibody, diluted (1:200) in the same blocking buffer (3 h, room temp.). The primary antibodies included rabbit polyclonal anti- VAChT (1:100, Sigma, St.
• Biotinylated secondary antibodies were donkey anti-rabbit (Chemicon) and donkey anti-goat (Jackson ImmunoResearch Laboratories, West Grove, PA), both used at 1:200 dilutions. Detection was with Fast Red substrate for anti- VAChT and ChAT antibodies and with Vectastain ABC peroxidase kit (Vector Laboratories, Buriingame, CA) for the anti-IL-l ⁇ antibody.
• Confocal microscopy was carried out using a Bio-Rad MRC 1024 confocal scanhead (Hemel Hempsted, Hertfordshire, U.K.) coupled to an inverted Zeiss Axiovert 135 microscope (Oberkochen, Germany) equipped with a Plan Apochromat 40X1.3 immersion objective. Fast Red was excited at 488 nm and emission was measured through a 580df32 interference filter (580 ⁇ 16 nm). Immunolabeled sections were scanned every 0.5 ⁇ m and projections analyzed using the Image Pro Plus 4.0 (Media Cybernetics, Silver Spring, MD) software.
• Image Pro Plus 4.0 Media Cybernetics, Silver Spring, MD
• Cholinesterase activity measurements Plasma samples were subjected to cholinesterase catalytic activity measurements [Ellman, G.L. et al. (1961) Biochem. Pharmacol. 7, 88-99] adapted to a multi-well plate reader. Acetylthiocholine (ATCh) hydrolysis rates were measured following prior incubation for 30 min with 5xlO" 5 M of the specific butyrylcholinesterase (BuChE) inhibitor tetraisopropylpyrophosphoramide, iso-OMPA. Total plasma cholinesterase activities were measured in the absence of inhibitors.
• Subjects of the memory study Ten male subjects participated in the study, which was approved by an independent ethics committee. Subjects recruitment as well as physical and psychiatric screening, were described in detail elsewhere [Reichenberg A. et al. (2001) id ibid.]. The current study involved a subset of the subjects included in the previous project, with serum AChE and working memory tests added. Interviews by experienced psychiatrists excluded the presence and the history of any axis I psychiatric disorder according to the DSM-IV [American Psychiatric Association (1994) Diagnostic and statistical manual for mental disorders, 4th ed. Washington DC]. Only subjects who successfully passed the screening procedure, and signed an informed consent form, were considered eligible to participate. Comprehensive assessment was performed, and involved each subject going through a number of physical and neuropsychological tests in a clinical research unit using a balanced, randomized, double-blind, cross-over design.
• Procedure for the memory tests All technical equipment, including the blood sampling device, was housed in a room adjacent to the sound-shielded experimental room. Every subject passed two 10 days apart testing sessions and spent the night before each experimental session in the research unit. A battery of neuropsychological tests, assessing memory, learning, and attention was given for adaptation upon their first arrival in the evening, minimizing subsequent practice effects [McCaffrey, R.J. and Lynch, J.K. (1992) Neuropsychol. Rev. 3:235-48]. Alternate versions of these tests were used in the experimental testing sessions.
• an intravenous cannula was inserted into an antecubital forearm vein for intermittent blood sampling and intravenous (i.v.) injection of endotoxin (0.8 ng Salmonella abortus equi endotoxin per Kg body weight) in one session or the same volume of 0.9% NaCl (saline) solution on the other occasion (placebo).
• endotoxin 0.8 ng Salmonella abortus equi endotoxin per Kg body weight
• saline 0.9% NaCl
• Salmonella abortus equi endotoxin Prepared for use in humans, this endotoxin was available as a sterile solution free of proteins and nucleic acids. The endotoxin preparation employed has proven to be safe in various studies of other groups [BurreU R. (1994) id ibid.] and in studies at the Max Planck Institute of Psychiatry, including more than 100 subjects since 1991 [Pollmacher T. et al. (1996) J. Infect. Dis. 174:1040-5].
• Plasma levels of AChE and its degradation product, cytokines and cortisol Blood was collected in tubes containing Na-EDTA and aprotinin and was immediately centrifuged. Plasma was aliquoted and frozen to -80°C AChE catalytic activity was measured as the capacity for acetylthiocholine (ATCh) hydrolysis in the presence of 1 x 10 -5 M tetraisopropylpyrophosphoramidate (iso-OMPA), a selective inhibitor of serum butyrylcholinesterase, BChE [Soreq H. and Glick D. (2000): Novel roles for cholinesterases in stress and inhibitor responses. In: Giacobini E.
• AChE-R mRNA and its protein product in vascular endothelial cells Fluorescent in situ hybridization and immunohistochemistry of AChE-R mRNA and AChE-R protein were performed and quantified as reported [Cohen (2002) id ibid.; Perry, C. et al. (2002) Oncogene 21:8428-8441] using paraffin- embedded tissue sections from surgically-removed biopsies of patients with or without clinical inflammation due to non-specific kidney vascuhtis or following kidney rejection.
• MALDI-TOF-MS analysis of immunolabeled proteins Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was employed in an attempt to identify the protein and peptide bands labeled by anti-AChE-R antibodies in blotted membranes. Proteolytic degradation of the gel - eluted peptide was performed using the endoprotease LysC from Achromobacterlyticus (Wako Chemicals, Inc., USA) at a substrate to enzyme ratio of 200:1. Digestion was carried out overnight in 0.05M Tris HCl, pH 9.0, containing 4M urea at 30°C
• Subjects were requested to repeat lists of digits with increased number of digits every two lists either in the correct order of presentation (forward condition-a.ssessm.ent of span), or in a reversed order (backward condition- assessment of working memory). The number of lists correctly repeated was counted. Attention was assessed using the Ruff 2&7 cancellation test [Ruff R. M. and Allen C. C (1996): Ruff 2&7 Selective Attention Test: Professional Manual. Psychological Assessment Resources Inc., Lutz, FL]: Subjects were instructed to mark either the digit 2 or the digit 7, which are randomly placed either between letters or between digits. The numbers of correct responses in a 5 minute trial were counted.
• Linear rank Wilcoxon test for two related samples was used for the analysis of AChE-R- and IL-l ⁇ -positive fractions of analyzed neurons, measured on at least 4 sections from each group. Differences were considered significant when & p value of ⁇ 0.05 or less was obtained using the SAS 8.0 software. Student's t test was used for analyzing the numbers and volume of VAChT-containing terminals in spinal cord sections.
• VAChT was predictably concentrated in cholinergic (C) terminals surrounding motoneurons [Weihe (1996) id ibid.], where it loads neural vesicles with ACh.
• VACh-T-labeled C-terminals were significantly smaller ( ⁇ 60 ⁇ m 3 ) under p.o. administration of 150 ⁇ g/Kg/day as compared to control sections (Fig. IB and IC, p ⁇ 0.01, Student's t test), perhaps reflecting changes in VAChT translocation into vesicles and/or VAChT stability.
• VAChT production is largely co-regulated with that of ChAT [Usdin, T. .et al. (1995) Trends Neurosci. 18, 218-224], since both are produced from one gene complex (the so called "cholinergic locus") [Erickson, J.D. et al. (1996) Prog. Brain Res. 109, 69-82].
• ChAT staining of C-terminals on motoneurons indeed presented similar changes to those observed for VAChT staining (data not shown).
• Fig. IE staining intensity of ChAT-positive partition cells
• Cholinesterase activities were measured in plasma samples taken during the second day of hENlOl administration.
• ATCh hydrolysis in plasma is largely due to serum BuChE, the primary serum cholinesterase encoded by a non- homologous mRNA which remained generally unchanged.
• plasma also includes a minor, but significant AChE activity [Zakut, H. et al. (1998) Cancer 61, 727-737], measurable following pre-incubation in the presence of 5xlO" 5 M of the BuChE-specific inhibitor, iso-OMPA.
• AChE activity increased, as compared with the values before treatment (pre-dose), within the 5 hr following the stressful oral gavage administration of 150 ⁇ g/Kg EN101 (Table 1), potentially reflecting increased production under handling.
• Example 3 EN101 effects on AChE-R and AChE-S mRNAs in monkey spinal cord neurons
• Paraffin-embedded sections of lumbar spinal cord from Cynomolgus monkeys treated for 7 days once daily with hENlOl were subjected to high resolution fluorescent in situ hybridization (FISH).
• FISH fluorescent in situ hybridization
• Fig. 2A Variant-specific FISH probes revealed AChE-S more than AChE-R mRNA labeling in numerous punctuate areas and longitudinal threads, possibly cross-sections and longitudinal sections through neuronal processes (Fig. 2B-2C).
• PET Positron Emission Tomography
• the blood-brain-barrier of primates may be more easily penetrated than that of rodents [Tomkins et al. (2001) Cell Mol. Neurobiol. 21: 675-91]. Nevertheless, this is the first demonstration of an organismal antisense response that affects primate CNS neurons.
• motoneurons 40 ⁇ m, 20-35% of total counted neurons, localized to motor nuclei in the ventral horn and intermediate zone
• medium-sized neurons 20-40 ⁇ m, about 60%, dispersed throughout the spinal cord, mainly in the ventral horn and intermediate zone
• small neurons 10-20 ⁇ m, 5-20%, located primarily in the dorsal horn.
• Reduced staining intensity suggested a certain antisense effect in motoneurons, as well, albeit with relatively limited efficacy. However, there was no discernable reduction in the total fractions of labeled large cell bodies by any treatment (p>0.100).
• AChE-S mRNA the number of large positive cell bodies remained unchanged, whereas positive small and medium sized neurons, were reduced by 50% and 20%, respectively under either low or high dose of hENlOl as compared to na ⁇ ve.
• the apparent dose-independence of changes in AChE-S mRNA is compatible with the hypothesis that these changes were not antisense driven, but could possibly reflect the effect of handling stress of shifting splicing from AChE-S to AChE-R [Kaufer (1998) id ibid.].
• Endotoxin administration produced a time-dependent decrease in plasma AChE activity, measured by quantifying the rate of ATCh hydrolysis in the presence of the butyrylcholinesterase (BChE) inhibitor iso-OMPA.
• BChE butyrylcholinesterase
• Fig. 5A Treatment-by-time interaction
• Saline administration caused no change in AChE activity, excluding the possibilities that it was induced by the injection stress or by circadian influences.
• the decline in hydrolytic activity could potentially reflect losses in the AChE protein.
• electrophoretically separated plasma proteins were immune-reacted with antibodies selective for the C- terminal peptide unique to AChE-R [Sternfeld et al.
• AChE-R cleavage product larger plasma samples (180 ⁇ g/lane) were resolved by electrophoresis. Protein bands that co-migrated with the bands labeled with anti AChE-R antibodies were cut out of the gel and subjected to MALDI-TOF-MS analyses. The elution product of the larger band was identified as being mainly composed of serum albumin (molecular weight, 69367), compatible with the assumption that AChE-R is only a minor component in this size fraction of human serum proteins. The shorter peptide eluted from the excised band, however, revealed a single peak with a molecular mass of 3613-3615.
• Figure 6 demonstrates the MALDI-TOF-MS profile of this eluted peptide.
• Peptide property calculations positioned the presumed proteolytic cleavage site 36 residues from the C- terminus of AChE-R, with a calculated mass of 3614. Under these assumptions, cleavage could occur between asparagine and arginine residues upstream to the AChE-R diversion site (Fig. 6).
• Example 8 Vascular endothelial cells produce AChE-R
• vascular endothelial cells displayed labeling with both AChE-R cRNA and anti AChE-R antibodies (Fig. 7A, 7B). Quantification of signal intensities revealed considerable similarities between AChE-R mRNA and AChE-R protein levels in patients with or without inflammatory vascuhtis, so that tissues with less pronounced mRNA labeling also displayed fainter protein labeling (Fig. 7C).
• Example 9 AChE-R cleavage is associated with cytokines secretion
• Endotoxin induced a transient, significant increase in the plasma levels of cortisol, TNF- ⁇ and IL-6 (Fig. 8A-8C), although at the employed dose it does not produce any significant effects on the subjective rating of physical or behavioral sickness symptoms [Reichenberg (2001) id ibid.].
• Cortisol levels increased during the first and second testing periods, TNF- ⁇ and IL-6 peaked during the first testing period and decreased thereafter and rectal temperature (not shown) peaked during the second period.
• F(2,16) 41.2, 10.6, 10.5, 3.2, respectively, all p ⁇ 0.05, by H-F].
• Example 10 AChE-R cleavage is associated with endotoxin-induced impairments in declarative memory
• Example 11 AChE-R cleavage association with improved working memory
• Fever is one of the consequences of higher levels of circulating pro- inflammatory cytokines.
• human synaptic AChE hAChE-S
• niAChE-R niAChE-R overexpressing females
• Example 13 Effects of Tacrine on LPS-induced IL-1 secretion in the hippocampus and IL-1 and TNF- ⁇ secretion in the serum.
• mice were deeply anesthetized with 24 ⁇ ig Nembutal per mouse, blood was taken by heart puncture and the hippocampus was excised and placed in tubes containing 500 ⁇ l of RPMI + 100 KIU aprotinin.
• the levels of IL-l ⁇ in the hippocampus Fig.
• Example 14 Effects of Rivastigmine on LPS-induced IL-1 secretion in the hippocampus and IL-1 and TNF- ⁇ secretion in the serum.
• Two types of stressful situations were investigated in the same subjects: Psychological stress — while waiting for a surgery (i.e., in the morning of the surgery day), and surgical stress — in the day after surgery.
• EN301 corresponds to mENlOl, defined herein as SEQ. ID. NO:2. This antisense ohgonucleotide is targeted to a sequence within exon 2 of mouse AChE exon 2 sequence.
• EN301 was produced by Microsynth, Switzerland, at relatively large quantities for animal tests. The treatment persisted for 3 consecutive days, and the mice were sacrificed on day 4. Brain was collected, flash frozen in liquid nitrogen and stored at -70°C
• the goal of the present experiment was to test for reduction in AChE gene expression under EN301 treatment, while ensuring that AChE-S mRNA levels are maintained reflecting sustained cholinergic neurotransmission.
• the ratio between AChE-S:common (S/Com) transcripts showed that in the EN301-treated brain, the S/Com ratio is significantly increased (from 0.65 to 0.98).
• RT-PCR data cannot be used as such for comparing the absolute quantities of the analyzed transcripts, because different primer pairs may function with different efficacies.
• these two tests point at the same direction (namely, that AChE-R but not AChE-S mRNA was reduced in the EN301-treated brains and that the relative concentration of AChE-S mRNA increased, albeit insignificantly, under treatment) supports the notion that this agent affects brain gene expression as well.
• EN301 treatment causes selective destruction of AChE-R mRNA in the EN301 treated brains while maintaining essentially unmodified AChE-S levels.
• EN301 does not necessarily have to cross the blood-brain barrier. Rather, by reducing the levels of peripheral AChE it would increase acetylcholine levels, suppressing the production by macrophages of pro- inflammatory cytokines e.g. IL-1 [Wang, H. et al. (2003) Nature 421, 384-8].
• IL-1 promotes AChE gene expression [Li et al. (2000) J. Neurosci. 20, 149-155]
• the peripheral pro-inflammatory cytokines are known to affect the brain [Pick et al. (2004) Annals NY Acad Sci. in press]
• such an effect will eventually reduce AChE-R levels in the brain as well.

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• 2003
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• 2004
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• 2005
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• 2007
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