EP0760666A1 - Utilisation de phosphorothioate oligonucleotidique pour la depletion du complement et la reduction de la pression sanguine - Google Patents

Utilisation de phosphorothioate oligonucleotidique pour la depletion du complement et la reduction de la pression sanguine

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Publication number
EP0760666A1
EP0760666A1 EP95920471A EP95920471A EP0760666A1 EP 0760666 A1 EP0760666 A1 EP 0760666A1 EP 95920471 A EP95920471 A EP 95920471A EP 95920471 A EP95920471 A EP 95920471A EP 0760666 A1 EP0760666 A1 EP 0760666A1
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EP
European Patent Office
Prior art keywords
oligonucleotide
complement
blood pressure
primate
administered
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95920471A
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German (de)
English (en)
Inventor
Wayne M. Galbraith
Sudhir Agrawal
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Aceragen Inc
Original Assignee
Hybridon Inc
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Filing date
Publication date
Application filed by Hybridon Inc filed Critical Hybridon Inc
Publication of EP0760666A1 publication Critical patent/EP0760666A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61P9/12—Antihypertensives

Definitions

  • This invention relates to the effect of synthetic oligonucleotides on in vivo complement activation and the results thereof . More specifically, this invention relates to methods of depleting complement , using synthetic oligonucleotides , which will lower blood pressure and trigger vasodilation.
  • the complement system is a cascading series of about 20 different plasma enzymes, enzyme precursors, regulatory proteins, and proteins capable of cell lysis and involved in the normal immune system response to foreign cells and in the abnormal immune system response to the individual's own cells. All of these proteins are normally present in the plasma and among the plasma proteins that leak out of capillaries into tissue spaces.
  • the enzyme precursors are normally inactive, but can be activated via two separate pathways: the classical pathway utilizing complement components Cl, C4, and C2; and the alternate pathway, via factors D, C3, and B. Both modes of activation lead to cleavage and activation of component C3. Fragment C3b split from C3, is necessary for the activation of the terminal complement components C5-C9. These form the membrane attack complex which, when inserted into cell membranes, brings about osmotic lysis of foreign cells, and in the case of autoimmune states, lysis of the affected organism's own cells.
  • Activation of the complement cascade results not only in cell lysis, but also in opsomization and phagocytosis of bacteria by macrophages and neutrophils, agglutination of invading organisms, neutralization of some viruses, chemotaxis of neutrophils and macrophages caused by C5a, activation of basophils and mast cells, and inflammation (Guyton, Textbook of Medical Physiology , .B. Sauders Co., Philadelphia (1991) pp. 374-384) .
  • Mast cell and basophil activation, followed by histamine release are triggered by fragments C3a, C4a, and C5a, which are enzymatically split off from C3, C4, and C5.
  • Rheumatoid arthritis is a chronic multisystem disease whose common clinical manifestation is persistent inflammatory synovitis of the peripheral joints resulting in proliferation of synovial cells and subsequent pannus formation, cartilage destruction, bone erosion, and ultimately joint deformity and loss of joint function. This disorder affects approximately 1% of the population of the United States and Europe as well as 0.2 to 0.4% of the Japanese population, with women being affected about three times more often than men.
  • Certain complement factors are potent vasodilators which can affect blood pressure and disorders related thereto such as hypertension.
  • Hypertension is a prevalent health problem in many developed countries. Many patients with hypertension die prematurely, with the most common cause of death being heart disease, stroke, and renal failure. Treatment typically consists of nondrug therapeutic intervention including stress relief, diet, weight reduction, regular aerobic exercise, and the administration of antihypertensive drugs including diuretics, antiadrenergic agents, vasodilators, calcium channel blockers, and angiotensin-converting enzyme (ACE) inhibitors.
  • ACE angiotensin-converting enzyme
  • the methods each involve the administration of an oligonucleotide phosphorothloate (a"PS- oligonucleotide”) having a sulphur substitution for one of the oxygens in at least one non- bridging phosphodiester intemucleotide linkage.
  • a PS- oligonucleotide oligonucleotide phosphorothloate having a sulphur substitution for one of the oxygens in at least one non- bridging phosphodiester intemucleotide linkage.
  • the oligonucleotide has only phosphorothloate intemucleotide linkages.
  • oligonucleotide is meant to encompass two or more nucleotides wherein the 5' end of one nucleotide and the 3' end of another nucleotide are covalently linked.
  • the oligonucleotides useful in the methods of the invention are from 2 to 50 nucleotides in length, with oligonucleotides having 6 to 50, and more preferably, 20 to 33 nucleotides in length being most useful in some embodiments.
  • the oligonucleotide has at least one deoxyribonucleotide or at least one ribonucleotide.
  • the oligonucleotides are chimeric, i.e., have a combination of both deoxyribonucleotides and ribonucleotides in any location or order in the molecule.
  • the oligonucleotides are modified.
  • modified oligonucleotide is used herein as an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups.
  • Preferable synthetic linkages include, in addition to phosphorothioates, linkages such as alkylphosphonates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, 2-0- methyls, and carboxymethyl esters.
  • linkages can be present anywhere in the oligonucleotide structure, and more than one type of linkage can be present in a single oligonucleotide (i.e., a hybrid oligonucleotide) .
  • modified oligonucleotide also encompasses oligonucleotides with a modified base and/or sugar.
  • a 3', 5' -substituted oligonucleotide is a modified oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) .
  • a modified oligonucleotide may also be a capped species.
  • unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
  • modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention are also considered herein as modified.
  • Oligonucleotides which are self-stabilized are also considered to be modified oligonucleotides useful in the methods of the invention (Tang et al. (1993) Nucleic Acids Res.
  • oligonucleotides comprise two regions: a target hybridizing region; and a self-complementary region having an oligonucleotide sequence complementary to a nucleic acid sequence that is within the self- stabilized oligonucleotide.
  • the oligonucleotide is administered as a bolus intravenous infusion at a constant rate of about 30 to 120 milligram oligonucleotide per kilogram recipient per hour (mg/kg/hr) .
  • the oligonucleotide is administered at a constant rate of about 30 mg/kg/hr, while in others, it is administered at about 40 mg/kg/hr.
  • Yet other methods of the invention require the administration of about 5 to 20 mg/kg oligonucleotide over a 10 minute period, while others require about 80 mg/kg over a 120 minute period.
  • the blood pressure of the recipient primate is measured after the administration of the oligonucleotide.
  • the blood pressure is measured 15 to 35 minutes after administration.
  • complement activity in a blood sample taken from the recipient primate is measured after the administration of the oligonucleotide.
  • the complement activity is measured 10 to 60 minutes after administration.
  • FIG. 1 is a mean arterial blood pressure profile of animals following intravenous administration of PS-oligonucleotide over a 10 minute period, beginning at time zero;
  • FIG. 2 is a mean arterial blood pressure profile of animals following intravenous administration of PS-oligonucleotide over a 120- minute period, beginning at time zero;
  • FIG. 3A is a graph showing the heart rate
  • FIG. 3B is a graph showing the heart rate
  • FIG. 3C is a graph showing the heart rate (_) and mean arterial pressure ( ⁇ ) of monkeys following administration of a single dose of PS- oligonucleotide at 1 mg/kg of the mammal over a 10 min. period;
  • FIG. 3D is a graph showing the heart rate (-) and mean arterial pressure ( ⁇ ) of monkeys following administration of a single dose of PS- oligonucleotide at 2 mg/kg of the mammal over a 10 min. period;
  • FIG. 3E is a graph showing the heart rate (-) and mean arterial pressure ( ⁇ ) of monkeys following administration of a single dose of The
  • GG oligonucleotide at 5 mg/kg of the mammal over a ten min. period;
  • FIG. 3F is a graph showing the heart rate (-) and mean arterial pressure ( ⁇ ) of monkeys following administration of a single dose of PS- oligonucleotide at 10 mg/kg of the mammal over a 10 min. period;
  • FIG. 3G is a graph showing the heart rate
  • FIG. 4 is a graph showing the level of complement (CH50) activity in animals following administration of various doses of PS- oligonucleotide intravenously over a ten minute period,-
  • FIG. 5 is a graph showing the level of complement (C5a) in animals following intravenous administration of various doses of PS- oligonucleotide over a 10 minute period;
  • FIG. 6A is a graph showing the level of complement (CH50) activity in human serum following the administration of various concentrations of PS-oligonucleotide.
  • FIG. 6B is a graph showing the level of complement (CH50) activity in serum from animals following administration of various concentrations of PS-oligonucleotide.
  • the present invention provides methods of depleting complement in a primate, which are useful, for example, in producing animal models that lack complement . Such animal models are of great value in examining the role of complement in various types of immune and other responses. Methods of depleting complement are also useful in slowing or inhibiting inflammation, and in reducing the lytic effects of various autoimmune disorders such as rheumatoid arthritis.
  • the present invention also provides methods of decreasing blood pressure and causing vasodilation in a primate. Such methods are useful in treating acute hypertension, a disease common in developed countries.
  • complement is depleted, blood pressure is reduced, and vasodilation is induced in a subject, by the administration of an oligonucleotide phosphorothloate having a sulphur substitution for one of the oxygens at least one non-bridging oxygen of a phosphodiester intemucleotide linkage (PS-oligonucleotide) .
  • PS-oligonucleotide oligonucleotide phosphorothloate having a sulphur substitution for one of the oxygens at least one non-bridging oxygen of a phosphodiester intemucleotide linkage
  • PS-oligonucleotides display resistance to enzymatic degradation, and have been studied extensively in the development of antisense oligonucleotide-based therapeutics (see e.g. , Zamecnik, Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wickstrom, E., Ed., Wiley-Liss, Inc., New York, New York, Vol. 1, 1991) .
  • PS- oligonucleotides have been used as antiviral agents (see, e.g. , Agrawal (1992) Trends Biotech.
  • anti-cancer agents see, e.g. , Ratajczak et al. (1991) Proc. Natl. Acad. Sci. (USA) 89:11823-11827; Bayever (1993) Antisense Res. Dev. 3:383) and anti-parasitic agents (see, e.g. , Rappaport et al. (1993) Proc. Natl. Acad. Sci. (USA) 89:8577-8580) in various in vitro model systems.
  • PS-oligonucleotides have been employed in regulating the expression of a number of cellular gene targets (see, e.g. , Stein et al . (1993) Science 261:1004) .
  • the PS-oligonucleotides used in the methods of the invention are composed of deoxyribonucleotides, ribonucleotides, or a combination of both, with the 5' end of one nucleotide and the 3' end of another nucleotide being covalently linked. These oligonucleotides are at least 6 nucleotides in length, but are preferably 10 to 50 nucleotides long, with 20 to 33mers being the most common. Some useful PS-oligonucleotides have one phosphorothloate linkage located between any two neighboring nucleotides in the molecule. Other PS-oligonucleotides have more than one phosphorothloate linkage between nucleotides scattered throughout the molecule or contiguously located. Yet others have only phosphorothloate linkages.
  • oligonucleotides useful in the methods of the invention may also be modified in a number of ways without compromising their ability to function in the methods of the invention.
  • the oligonucleotides may contain, in addition to at least one phosphorothloate linkage, an intemucleotide linkage other than a phosphorothloate inte ucleotide linkage between the 5' end of one nucleotide and the 3' end of another nucleotide.
  • the 5' nucleotide sulfur in the case of a phosphorothloate
  • Examples of such chemical groups include alkylphosphonates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters .
  • modifications include those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins .
  • modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3', 5' -substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) .
  • Other modified oligonucleotides are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide.
  • Such modifications can be at some or all of the intemucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.
  • oligonucleotides include those that are self-stabilized, as described in Tang et al. (Nucleic Acids Res. (1993) 21:2729-2735) .
  • Such oligonucleotides have a target hybridizing region and a self-complementary region having an oligonucleotide sequence complementary to a nucleic acid sequence that is within the self- stabilized oligonucleotide.
  • nucleotides can be covalently linked using art-recognized techniques such as phosphoramidate, H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal et al . (1987) Tetrahedron. Lett. 28 : (31) :3539-3542) ; Caruthers et al. (1987) Meth. Enzymol.
  • Oligomeric phosphorothloate analogs can be prepared using methods well known in the field such as methoxyphosphoramidite (see, e.g. , Agrawal et al . (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083) or H-phosphonate (see, e.g. , Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry.
  • the synthetic methods described in Bergot et al . J. Chromatog. (1992) 559:35-42) can also be used.
  • oligonucleotide examples include those listed below in TABLE 1 and set forth in the Sequence Listing as SEQ ID NOS:1-6.
  • oligonucleotides can have any nucleotide sequence, as the effects caused by the administration of these oligonucleotides is not sequence specific.
  • the "GG" oligonucleotide (SEQ ID N0:1) is complementary to the gag initiation codon of HIV-1 (Agrawal and Tang (1992) Antisense Res. Dev. 2:261) .
  • the other five oligonucleotides are phosphorothioates varying in length from 20 to 33 nucleotides.
  • the 25mers tested were a mixture of 4 24 25mer random sequences (25mer random) .
  • the 25-mer random was synthesized by using a mixture of A, C, G, and T for each coupling during synthesis.
  • the oligonucleotides are administered via intravenous injection to the subject in the form of a therapeutic formulation which contains at least one PS-oligonucleotide as described above, along with a physiologically acceptable carrier.
  • a "physiologically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art . Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi.
  • the carrier can be a solvent or dispersion medium.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents. Prolonged absorption of the injectable therapeutic agents can be brought about by the use of the compositions of agents delaying absorption.
  • the therapeutic formulation is injected intravenously in one bolus, which may be repeated at various time intervals as needed.
  • the rate of injection is dependent on the amount of oligonucleotide being administered, with 30 to 120 mg per kg weight of the recipient per hour being in the acceptable range.
  • a bolus administration of 5 to 20 mg/kg over a 10 minute period, or 80 mg/kg over a 120 minute period results in the lowering of blood pressure, vasodilation, and depletion of complement.
  • the blood pressure of the primate is measured after it has been treated with the oligonucleotide and a drop in blood pressure is ascertained. This may be accomplished by any known means of measuring blood pressure.
  • blood pressure may be measured by placing a catheter in the femoral artery or by extracorporeal monitoring with a blood pressure gauge. As the largest decrease in blood pressure is seen 15 to 35 minutes after administration of the oligonucleotide to the primate, this time period is preferred for taking such measurements.
  • complement activity in the blood or serum of the primate is measured after it has been treated with the oligonucleotide and a decrease in complement activity is ascertained.
  • This may be accomplished by any known means of assaying for complement components of activity.
  • complement CH50 can be measured by complement-dependent lysis of sheep red blood cells as described in Kabat et al . (Expt. Immunochem. (1961) Charles C. Thomas, New York)
  • C5a can be measured by radioimmunoassay. As the largest depletion of complement is seen 10 to 60 minutes after administration of the oligonucleotide to the primate, this time period is preferred for taking such measurements.
  • PS-oligonucleotides have been found to be well tolerated in mice (Agrawal, Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS , W. Liss, New York, (1991) p.143) and rats; however, in monkeys, acute hemodynamic toxicity has been observed under certain circumstances. There is a recent report of hypotension and death in the Rhesus monkey following bolus administration of a PS- oligonucleotide directly into the aorta (Cornish (1993) Pharm. Comm. 3:239-247) . This invention demonstrates the effects of the administration of PS-oligonucleotides of varying lengths and sequences in primates.
  • SEQ ID NO:l was administered to primates over a 10 minute time interval at doses of 0 (saline) or 1.25 mg/kg, there was no detectable effects on mean arterial blood pressure (FIG. 1) or heart 0 rate. On the other had, a 10 minute infusion of 5 mg/kg produced a transient increase in mean blood pressure by the end of the infusion period (FIG. . _ .1) , followed by a more prolonged decreased pressure. A dose of 20 mg/kg of GG over 10 5 minutes produced a similar transient blood pressure increase, followed by a more pronounced and more prolonged hypotension.
  • Concentrations of serum complement CH50 decreased at doses greater than or equal to 10 mg/kg, beginning within 5 minutes of the start of treatment (FIG. 4) .
  • the blood samples were drawn at 10 minutes prior to dosing and at 2, 5, 10, 20, 40, 60 minutes and 25 hours post-dosing and analyzed for level of CH50 complement.
  • C5a split products of complement increased markedly, beginning within 2 minutes of infusion at doses greater than or equal to 5 mg/kg; the higher the dose, the earlier the appearance of increased C5a (FIG. 5) . At doses less than or equal to 2 mg/kg, no changes were observed.
  • hypotension induced by intravenous oligonucleotide is clearly dose- and infusion ratedependent.
  • the dose-response curve can be marked shifted to the right by decreasing the rate of oligonucleotide infusion.
  • a dose of 80 mg/kg over 10 minutes (or 30 mg/kg/hr) produces a similar blood pressure response as a dose of 5 mg/kg over 10 minutes (or 30 mg/kg/hr) .
  • the effects of the oligonucleotide on hemodynamics appear to the mediated by peripheral vascular changes since there is no evidence of direct effects on the heart.
  • the oligonucleotide with SEQ ID N0S: 1 , 3, 5, and 6 were prepared, as well as a 25-mer mixture of 4 24 sequences (25-mer random) .
  • the 25-mer random was synthesized by using a mixture of A, C, G, and T for each coupling during synthesis (Lisziewicz et al. (1993) Proc. Natl. Acad. Sci. (USA) 90:3860) .
  • GG SEQ ID N0:1 or other PS-oligonucleotide was dissolved in normal saline and infused intravenously via a cephalic vein catheter using a programmable infusion pump. In all cases, the concentration of GG was such as to allow the dose to be delivered at a rate of 0.42 ml/min.
  • GG doses of 0, 0.5, 1, 2, 5, 10, and 20 mg/kg were administered to 2 animals each over a 10 minute infusion period.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • SEQUENCE DESCRIPTION SEQ ID NO:1 :
  • MOLECULE TYPE CDNA/RNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES

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Abstract

L'invention concerne des procédés de réduction de la pression sanguine, de stimulation de la vasodilatation, et de déplétion du complément chez un primate. Ces procédés consistent à administrer un oligonucléotide au primate, et à mesurer la réduction de la pression sanguine ou de l'activité du complément. L'oligonucléotide administré présente une longueur de 2 à 50 nucléotides et au moins une liaison internucléotidique phosphorothioate.
EP95920471A 1994-05-27 1995-05-19 Utilisation de phosphorothioate oligonucleotidique pour la depletion du complement et la reduction de la pression sanguine Withdrawn EP0760666A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US25085394A 1994-05-27 1994-05-27
US250853 1994-05-27
PCT/US1995/006161 WO1995032719A1 (fr) 1994-05-27 1995-05-19 Utilisation de phosphorothioate oligonucleotidique pour la depletion du complement et la reduction de la pression sanguine

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EP0760666A1 true EP0760666A1 (fr) 1997-03-12

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EP (1) EP0760666A1 (fr)
JP (1) JPH10501224A (fr)
AU (1) AU2591495A (fr)
CA (1) CA2191192A1 (fr)
WO (1) WO1995032719A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6232296B1 (en) 1999-09-30 2001-05-15 Isis Pharmaceuticals, Inc. Inhibition of complement activation and complement modulation by use of modified oligonucleotides
ES2911034T3 (es) 2006-08-08 2022-05-17 Univ Bonn Rheinische Friedrich Wilhelms Estructura y uso de oligonucleótidos 5' fosfato
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
EP2508530A1 (fr) 2011-03-28 2012-10-10 Rheinische Friedrich-Wilhelms-Universität Bonn Purification d'oligonucléotides triphosphorylés au moyen d'étiquettes de capture
EP2712870A1 (fr) 2012-09-27 2014-04-02 Rheinische Friedrich-Wilhelms-Universität Bonn Nouveaux ligands de RIG-I et procédés pour les produire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9532719A1 *

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JPH10501224A (ja) 1998-02-03
AU2591495A (en) 1995-12-21
WO1995032719A1 (fr) 1995-12-07

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