ANTISENSE DRUGS AND MYASTHENIA

This page last updated on:  April 09, 2005

A new drug for the treatment of myasthenia gravis has recently been developed and is under test in Israel and the United Kingdom.   This webpage has been setup to track the most recently published information related to this development.  To add information please contact the Webmaster.

Definition:  Antisense Drug

A synthetic segment of DNA or RNA that locks onto a strand of DNA or RNA with a complementary sequence of nucleotides. Antisense drugs are designed to block viral genetic instructions, marking them for destruction by cellular enzymes, in order to prevent the building of new virus or the infection of new cells."

An online explanation of antisense drugs written before the latest discovery of Ester Neuroscience from a group discussion provides more details..

new.gif (1556 bytes)ISRAEL21c has an article which includes mention of Phase II Trials of Monarsen to begin by the end of March 2004 in the United States and Europe.  December 14, 2003

new.gif (1556 bytes) The United States FDA approves EN101 for Orphan Drug Status.  Nov. 24, 2003.
http://www.esterneuro.com/RD_regulatory.html
The drug, EN101, has formally been named Monarsen.

Presented at "Joint congress Association of British Neurologists and the Neurological Association of South Africa, University of Cape Town, 29 January – 1 February 2003"

081 A PHASE 1B SAFETY, EFFICACY, AND PHARMACOKINETIC STUDY OF THE ANTISENSE OLIGONUCLEOTIDE EN101 IN PATIENTS WITH MYASTHENIA GRAVIS
D.H. McKee, J.D. Sussman. Greater Manchester Neurosciences Centre, Hope Hospital, Salford, UK, and University of Manchester UK (both authors)

We present results from the first clinical trial of EN101, a synthetic antisense oligonucleotide directed against acetylcholinesterase mRNA, in human subjects with stable myasthenia gravis requiring daily pyridostigmine for symptom control.

Patients stopped pyridostigmine for 18 hours, with subsequent deterioration in myasthenic symptoms, measured by the Quantitative Myasthenia Gravis score (QMG). Escalating oral doses of EN101 were given until a significant improvement in QMG occurred. Once daily dosing continued for three days, followed by a washout period during which the subjects’ clinical condition deteriorated and pyridostigmine was reinstituted. Following treatment with EN101 subjects showed both statistically and clearly clinically significant changes in QMG scores, sustained for up to 28 hours following final dosing. No serious adverse events were observed. By contrast with previous treatment with pyridostigmine, cholinergic side effects such as abdominal cramps and diarrhoea were conspicuous by their absence. Conversely, dryness of the mouth was reported in the majority.

Despite theoretical difficulties inherent in antisense oligonucleotide therapeutics, EN101 appears to be powerfully effective in reversing myasthenic symptoms, with significant advantages over cholinesterase inhibitors, particularly in the context of an acute deterioration. The results of this study justify proceeding to a double blind, randomised controlled trial.


PRESS RELEASE:  APRIL 1, 2003

Oral Antisense Therapy Successful in Ester Neuroscience Trial for Myasthenia Gravis
Tuesday April 1, 9:05 am ET

HONOLULU & HERZLIYA, Israel--(BUSINESS WIRE)--April 1, 2003--Ester Neuroscience announced today that the results of a successful Phase Ib trial using the company's EN101 drug for myasthenia gravis were presented at a special Late Breaking Science session of the American Academy of Neurology.

The breakthrough study demonstrates for the first time effective and safe use of an orally-administered antisense therapy for a neurological condition.

Prof. Zohar Argov of Hadassah University Hospital, the principal investigator reported that 15 out of 16 patients showed a clear symptomatic improvement due to EN101 without any adverse events.

"EN101 appears to be effective in reversing symptoms in patients with stable MG. EN101 has potential advantages over conventional cholinesterase inhibitors with respect to dosing, specificity, side-effect profile, duration of efficacy and treatment regimen," said Prof. Argov.

Late Breaking Science sessions of the American Academy of Neurology are devoted to neuroscience research of an extraordinary nature, which warrant expedited presentation. The trial was carried out at sites in Israel and in the U.K.

Dr. Jon Sussman, a neurologist at the Greater Manchester Neuroscience Centre in the U.K. a leading expert in the treatment of myasthenia gravis and lead investigator at the U.K.site, commented: "We were very impressed with the striking improvement in the condition of our patients. EN101 even enabled some patients with limited mobility to regain their ability to stand and walk without aids."

EN101 is the lead compound in Ester's disease-modifying platform technology for the pre-expression control of a specific variant of the AChE protein, which is applicable to a wide range of neurological disorders.

"The results of the study suggest that our oral anti-sense platform technology may have wide applications in the treatment of many other PNS and CNS disorders such as Alzheimer's disease and head injury," said Dr. Eli Hazum, CEO of Ester Neuroscience.

The technology is based on balancing cholinergic transmission via controlled modulation of the company's novel target, a stress-response variant of acetylcholinesterase (AChE). AChE is an enzyme that degrades the neurotransmitter acetylcholine. EN101 selectively inhibits the production of the target at the critical stage of its biosynthesis thereby allowing an effective treatment, while minimizing side effects and substantially improving upon the short-duration palliative relief currently observed with conventional inhibitors.

Oral delivery is a critical attribute that has been long sought after by antisense drug developers. Oral delivery is expected to improve patient compliance as it eliminates the need for daily or more frequent injections.

Myasthenia gravis (MG) is a chronic and debilitating disease characterized by muscle weakness that affects about 15 persons per 100,000.

Ester Neurosciences Ltd. is a clinical stage biotech company committed to the discovery and development of novel therapeutic products for the treatment of neurological disorders.

Ester Neuroscience is the developer and manufacturer of EN101, an antisense drug.

A publication by Ester Neurosciences in the June 2003 issue of DDT  (see link above).

A June 17, 2003 presentation at the 13th European Neurological Association meeting in Turkey.

http://biolchem.huji.ac.il//soreq.html

One of the primary researchers in Israel is Professor Hermona Soreq.  The above webpages describe some of her related work..

http://www.mfa.gov.il/mfa/go.asp?MFAH0ndw0

The above link is to an Israel government website talking about the Ester EN101 development.  The statement is at the bottom of the above webpage.

http://www.esterneuro.com/News/news1.html

The above link is to a February 21, 2002 Press Release announcing the approval for Phase 1b testing.

http://www.biospace.com/ccis/news_story.cfm?StoryID=6698615&full=1

http://www.biospace.com/ccis/news_story.cfm?StoryID=7598615&full=1

http://www.pharmacopeia.com/corp/cnews/pr/pr20011023.html

http://web.njit.edu/~nm3/biol601/poster.ppt   A single page describing the antisense problems.

The following are abstracts of antisense research copied from PubMed

1.  FASEB J 2003 Feb;17(2):214-22
The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis.

Brenner T, Hamra-Amitay Y, Evron T, Boneva N, Seidman S, Soreq H.

Department of Neurology, Hadassah University Hospital and Hebrew University Hadassah Medical School, Jerusalem, Israel 91120.

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare "readthrough" acetylcholinesterase variant AChE-R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE-R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE-R in blood and muscle yet leaves unaffected the synaptic variant AChE-S. Whereas animals treated with placebo or conventional anticholinesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA-targeted therapeutics for chronic cholinergic malfunctioning.

2.  Isr Med Assoc J 2000 Jul;2 Suppl:81-5
Anti-sense approach to anticholinesterase therapeutics.

Soreq H, Seidman S.

Department of Biological Chemistry, Hebrew University, Jerusalem, Israel. soreq@cc.huji.ac.il

The acetylcholine-hydrolyzing enzyme, acetylcholinesterase, is the molecular target of approved drugs for Alzheimer's disease and myasthenia gravis. However, recent data implicate AChE splicing variants in the etiology of complex diseases such as AD and MG. Despite the large arsenal of anti-AChE drugs, therapeutic inhibitors are primarily targeted towards an active site shared by all variants. In contrast, anti-sense oligonucleotides attack unique mRNA sequences rather than tertiary protein structures. AS-ODNs thus offer a means to target gene expression in a highly discriminative manner using very low concentrations of drug. In light of the likely role(s) of specific AChE variants in various diseases affecting cholinergic neurotransmission, the potential contribution that anti-sense technology can make towards improved approaches to anti-AChE therapeutics deserves serious attention.
3.  J Dermatol Sci 1999 Nov;21(3):157-64
Complementary peptides against the major epitope in the NC16A domain of BP180 show no specificity as vaccines to bullous pemphigoid.

Nie Z, Garrod DR, Chan LS, Hashimoto T.

Department of Dermatology, Kurume University School of Medicine, Fukuoka, Japan.

A stretch of 14 amino acids (542-555) (MCW-1) in the NC16A domain of BP180 has been shown to be an immunogenic and pathogenic epitope for bullous pemphigoid (BP). Therefore, it provides an excellent target for treatment through a complementary peptide approach, which has been established in other autoimmune diseases, including experimental autoimmune myasthenia gravis. We examined two synthetic complementary peptides BP3CP5 and BP5CP3 against this region. These peptides were derived, respectively, by reading the antisense RNA of this region of BP180 in 3'-5' and 5'-3' directions. We found evident complementarities in hydropathic scores between MCW-1 and both complementary peptides. However, by enzyme-linked immunosorbent assay (ELISA), the complementary peptides BP3CP5 and BP5CP3 did not bind to either synthetic peptide BPNP or glutathione-S-transferase (GST) fusion proteins BP180NC16a and GST-BP-1050. BPNP, BP180NC16a and GST-BP-1050 cover the MCW-1 region of BP180 and were used as the natural peptides in this study. In addition, neither BP3CP5 nor BP5CP3 blocked the reaction between BPNP and anti-BPNP antibody, nor did they block immunofluorescent staining of the basement membrane zone by BP sera. Pre-incubation with BP3CP5 and BP5CP3 did not block the binding of BP sera to the BP18NC16a fusion protein in immunoblotting. Furthermore, rabbit antisera raised against BP3CP5 and BP5CP3 did not bind BP sera in ELISA. Pre-incubation with these rabbit antisera did not inhibit or reduce the binding of BP sera to the autoanltigen in either imnmunoblotting or immunofluorescence. Thus, we concluded that complementary peptides against this particular epitope in BP180 NC16A domain showed no specificity as vaccines to BP, although this approach should be tried for other epitopes in various autoimmune bullous diseases.
4. Ann N Y Acad Sci 1993 Jun 21;681:83-96
Nicotinic neuronal acetylcholine receptor alpha-3 subunit transcription in normal and myasthenic thymus.

Mihovilovic M, Hulette C, Mittelstaedt J, Austin C, Roses AD.

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.

Thymic transcription of the alpha-3 subunit of the AChR was studied through sequencing and PCR analysis of thymic cDNA clones, Northern blotting, and ribonuclease protection assays. This analysis revealed at least three, 3' end sequence variants for the alpha-3 subunit as well as a variant that results from the alternative splicing of an antisense 122 bp Alu sequence between exons 5 and 6 of the normal transcript. The spliced Alu sequence not only shifts the exon 6 reading frame but also carries an in-frame stop codon. If translated, this variant transcript would produce a truncated peptide lacking the fourth transmembrane domain of the subunit and carrying a carboxy terminus dodecapeptide not found in any other known AChR subunit sequence. The putative variant subunit may lack biological activity and should differ antigenically from its normal counterpart. In comparing the normal, the MG hypertrophic, and the MG thymoma for transcription of the alpha-3 subunit and its 122 bp variant, it was found that there were no qualitative or quantitative changes in alpha-3 transcript expression in the MG hypertrophic thymi. Thymomas, however, showed an overall decrease in alpha-3 transcription and a comparative increase in beta-amyloid precursor transcription. The decrease in the levels of alpha-3 transcription in thymomas may be related to the proliferation of thymic epithelial cells.
5. Cell Immunol 1991 Nov;138(1):79-93
Synergistic induction of interleukin-6 production and gene expression in human thymic epithelial cells by LPS and cytokines.

Cohen-Kaminsky S, Delattre RM, Devergne O, Rouet P, Gimond D, Berrih-Aknin S, Galanaud P.

CNRS URA-1159, Hopital Marie Lannelongue, Le Plessis Robinson, France.

We examined the ability of LPS and several cytokines (TNF-alpha, IL-1-beta, IFN-gamma, IL-4) to modulate IL-6 production by cultured human thymic epithelial cells (TEC). IL-6 activity was measured by the hybridoma growth factor biological activity. Moderate but detectable IL-6 activity was spontaneously produced in the presence of serum proteins. LPS as well as the cytokines TNF-alpha and IL-1-beta was a potent inducer of IL-6, increasing, respectively, IL-6 levels by 9-, 28-, and 75-fold (mean values) while IL-4 and IFN-gamma provoked no significant effect. Interestingly, clearly different kinetics were observed for IL-6 induction by the various activation agents, the maximal effect being reached at 24, 48, and 72 hr, respectively for LPS, TNF-alpha, and IL-1-beta. Moreover, a synergistic effect of TNF-alpha and either LPS or IL-1-beta was observed. Indeed, TEC incubated with the cytokines in combination at optimal doses produced 5- to 170-fold more IL-6 than TEC stimulated with the cytokines individually. Neutralizing anti-IL-6 polyclonal and monoclonal antibodies completely blocked hybridoma proliferation stimulating activity of TEC supernatants; thus, implying that this activity is essentially due to IL-6. In situ hybridization analysis of cytocentrifuged TEC with an mRNA antisense probe specific for human IL-6 and labeled with 35S demonstrated that up to 90% of TEC could be induced to express the IL-6 gene. Computer-aided quantification of IL-6 mRNA levels indicated that upon stimulation with TNF-alpha combined to LPS, both the numbers of cells expressing IL-6 mRNA and the amounts of cytoplasmic IL-6 mRNA per cell were increased. Taken altogether these results demonstrate that LPS and/or cytokines can modulate and synergistically stimulate IL-6 production. In addition to a possible role in regulating normal thymic T cell activation, the IL-6 produced by TEC could be of pathophysiological relevance in disregulated situations such as in hyperplastic thymuses from patients with myasthenia gravis.