EP1401868A2 - Modulateurs de canaux ioniques - Google Patents

Modulateurs de canaux ioniques

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Publication number
EP1401868A2
EP1401868A2 EP02748981A EP02748981A EP1401868A2 EP 1401868 A2 EP1401868 A2 EP 1401868A2 EP 02748981 A EP02748981 A EP 02748981A EP 02748981 A EP02748981 A EP 02748981A EP 1401868 A2 EP1401868 A2 EP 1401868A2
Authority
EP
European Patent Office
Prior art keywords
icmm
functional equivalent
ion channel
peptide
polypeptide
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Withdrawn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
EP02748981A
Other languages
German (de)
English (en)
Inventor
Patricia Anne Nuttall
Miles Andrew Nunn
Peter Takac
Olga Pechanova
Petra Rajska
Milan Kozanek
Norbert Vrbjar
Maria Kazimirova
Milan Labuda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evolutec Ltd
Original Assignee
Evolutec Ltd
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Filing date
Publication date
Application filed by Evolutec Ltd filed Critical Evolutec Ltd
Publication of EP1401868A2 publication Critical patent/EP1401868A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to ion channel modulators.
  • the invention relates to ion channel modulators that are derived from haematophagous arthropods.
  • the invention also relates to the use of ion channel modulators from haematophagous arthropods in the treatment and prevention of certain diseases and conditions in mammals,
  • Ion channels are proteins which allow ions to cross the lipid bilayer of cell membranes. They exert control over the movement of ions by being either open or closed. Ion channel proteins can be divided into two groups. The first group of ion channel proteins form narrow hydrophilic pores across the membrane, allowing the passive movement of small inorganic ions. The second group of ion channel proteins can be coupled to a source of energy to promote active transport of ions across the cell membrane. Through a combination of passive and active transport using ion channels, cells can generate ionic concentration differences across the lipid bilayer of cell membranes. The electrical gradient thus generated can be used by cells in their signalling and control systems.
  • Ion channels show selectivity with respect to the ions to which they are permeable and respond to different opening and closing stimuli (eg ligand gated or voltage gated channels).
  • Modulators of ion channels can be divided into blocking agents which can only close channels and modulators which can either open or close them (Hille, 1992)
  • the present invention is particularly concerned with the modulation of ion channels in vascular smooth muscle cells and cardiac muscle cells. In such cells, propagation of an action potential over the membrane surface of the cell is associated with contraction of the cell. This effect is usually achieved by a small change in the resting membrane potential triggering the opening of a voltage gated ion channel which then permits the influx of large quantities of positively charged ions with a consequent change in the electrostatic conformation of the cell.
  • the small change in the resting membrane potential is achieved through active transport of Na + and K + ions by the Na + - + pump.
  • This pump depends on an energy source to drive the process and this is provided by the catalysis of ATP by the enzyme Na,K-ATPase.
  • the contraction of smooth muscle, including cardiac smooth muscle is dependent on a transient increase in cytoplasmic Ca ++ concentration, such ions coming from two sources: influx from the extracellular compartment by opening of inward Ca ++ channels and release of intracellular Ca ++ ions from the cisternae of the sarcoplasmic reticulum. Calcium channel blocking agents that inhibit the influx of extracellular Ca ++ ions will therefore tend to relax most smooth muscle cells including cardiac cells.
  • the force of contraction of cardiac smooth muscle is termed the inotropic state and, therefore, agents which relax the resting state of cardiac smooth muscle cells, such as most calcium channel blockers, are negatively inotropic. Conversely agents that increase the force of contraction of cardiac smooth muscle are positively inotropic.
  • Calcium channel blockers are used therapeutically to relax vascular smooth muscle thereby increasing the diameter of blood vessels ('vasodilation') in order to lower blood pressure or increase regional blood flow (eg via the coronary arteries).
  • the coincidental lowering of the force of cardiac smooth muscle contraction is an inherent disadvantage of most calcium channel blocking agents and precludes their use in conditions in which the functioning of the heart is already compromised. Such conditions include congestive heart failure, cardiomyopathies, septicaemia and following myocardial infarction. (Alberts et al, 1998; Camm, 1996a; Hume et al, 1998; Akera et al, 1998).
  • agents which possess the desirable qualities of vasodilators but which are not negatively inotropic would be positively inotropic.
  • drugs having a positive inotropic effect may be used.
  • Positively inotropic drugs such as digoxin frequently work through inhibition of sodium-potassium ATPase (Camm, 1996b).
  • positively inotropic drugs which include digoxin, dopamine, isopraline and phopshodiesterase inhibitors such as enoximone and milrinone, often have other, undesirable, effects on the heart such as the induction of arrhythmia.
  • an ion channel modulator molecule derived from an haematophagous arthropod, or a functional equivalent thereof.
  • ion channel modulator molecule is meant any molecule that modulates the activity of an ion channel.
  • the ICMMs are proteins or peptides. However, they may also be non-peptidic derivatives. Preferably, non-peptidic derivatives are small organic molecules.
  • ion channel is meant any transmembrane protein or transmembrane protein complex present in the cell that allows the movement of particular ions from one side of a cell membrane to the other.
  • the ICMMs or functional equivalents of the present invention modulate the activity of ion channels that allow the movement of ions across the cell membrane by either active or passive transport.
  • ion channels that allow the movement of ions by active transport include calcium ATPase and sodium-potassium ATPase.
  • ion channels that allow movement of ions by passive transport include calcium channels, sodium channels and potassium channels. These channels tend to be of particular relevance to disease, such as cardiac disease.
  • the ICMMs or functional equivalents of the invention may modulate more than one ion channel or class of ion channel. Such modulators may allow the simultaneous modulation of ion channels of different classes.
  • ICMMs may permit vasodilation by means of blockade of one or more calcium channels whilst also causing positive inotropism by means of inhibition of the sodium-potassium ATPase channel.
  • the ICMMs or functional equivalents of the present invention may modulate the activity of ion channels by either inhibiting or promoting the activity of ion channels.
  • the ICMMs or functional equivalents of the invention inhibit the activity of ion channels.
  • an ion channel that the ICMMs or functional equivalents of the present invention may inhibit is the sodium-potassium ATPase ion channel.
  • ICMMs or functional equivalents of the invention inhibit the activity of ion channels by binding to them.
  • the ICMMs or functional equivalents may be vasodilators.
  • Vasodilation may be promoted by the ICMMs functional equivalents of the invention, for example, through blockade of calcium channels or by nitric oxide donation.
  • the ICMMs or functional equivalents may be vasodilators of coronary vessels, peripheral vessels or both types of vessel.
  • the ICMMs, or functional equivalents of the invention act as vasodilators of both coronary vessels and peripheral vessels.
  • the ICMMs or functional equivalents of the invention preferably do not induce a negative inotropic effect in cardiac smooth muscle.
  • the ICMMs or functional equivalents induce a positive inotropic effect.
  • the ICMMs or functional equivalents of the invention may function to prolong the action potential of muscle cells.
  • the ICMMs or functional equivalents of the invention prolong the action potential of cardiomyocyte cells. This property may be important in prevention or treatment of arrhythmias such as are sometimes associated with the use of other positively inotropic drugs.
  • the ICMMs or functional equivalents of the present invention are derived from haematophagous arthropods.
  • haematophagous arthropod includes all arthropods that take a blood meal from a suitable host, and includes insects, ticks, lice, fleas and mites.
  • the ICMMs or functional equivalents of the present invention are derived from horseflies of the Tabanidae family. More preferably, they are derived from horseflies of the Hybomitra, Heptatoma, Chrysops, Haematopota and Tabanus genera. Most preferably, they are derived from the horsefly Hybomitra bimaculata.
  • ICMMs of the invention that retain the ability to modulate ion channels.
  • Functional equivalents of the ICMMs of the present invention thus include natural biological variants (e.g. allelic variants or geographical variants within the species from which the ICMMs are derived).
  • Variants of the proteinaceous ICMMs of the invention also include, for example, mutants containing amino acid substitutions, insertions or deletions from the wild type sequence.
  • Variants with improved function from that of the wild type sequence may also be designed through the systematic or directed mutation of specific residues in the protein sequence. Improvements in function that may be desired will include greater specificity for the target ion channel or greater affinity for the target ion channel.
  • the term "functional equivalent” also refers to molecules that are structurally similar to the proteinaceous ICMMs of the present invention or that contain similar or identical tertiary structure, particularly in the environment of the active site. Such functional equivalents may thus be derived from natural proteinaceous ICMMs or they may be prepared synthetically or using techniques of genetic engineering. In particular, synthetic molecules that are designed to mimic the tertiary structure or active site of the natural proteinaceous ICMMs of the invention are considered to be functional equivalents.
  • the term “functional equivalent” also includes fragments of the proteinaceous ICMMs of the present invention, fragments of variants of the proteinaceous ICMMs and fragments of structurally similar molecules, provided such fragments retain the ability to modulate ion channels.
  • protein fragments according to the invention comprise the amino acid sequence psggrrs. This amino acid sequence may be the active site of proteinaceous ICMMs, although the Applicant does not wish to be bound by this theory.
  • the term “functional equivalent” also refers to homologues of the proteinaceous ICMMs. By “homologue” is meant a protein exhibiting a high degree of similarity or identity to the amino acid sequence of a natural proteinaceous ICMM.
  • similarity is meant that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • identity is meant that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
  • homologues possess greater than 50% identity with the sequence of the natural protein. More preferably, homologues according to the invention show greater than 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the sequence of the natural protein, as aligned using, for example, the GCG suite of programs (Wisconsin Package Version, 10.1, Genetics Computer Group (GCG), Madison, Wise.) or the ExPASy (ExpertProtein Analysis System) proteomics server of the Swiss Insitute of Bioinformatics.
  • GCG suite of programs Wiconsin Package Version, 10.1, Genetics Computer Group (GCG), Madison, Wise.
  • ExPASy ExpertProtein Analysis System
  • Such homologues may include proteins in which one or more of the amino acid residues are substituted with another amino acid residue and such substituted amino acid residue may or may not be a naturally occurring amino acid.
  • the term "functional equivalent” also includes derivatives of the present invention.
  • Such derivatives may include one or more additional peptides or polypeptides fused at the amino- or carboxy- terminus of the proteinaceous ICMMs, fragments or homologues.
  • the purpose of the additional peptide or polypeptide may be to aid the detection, expression, separation or purification of the protein or it may endow the protein with additional properties, as desired.
  • useful fusion partners include beta-galactosidase, glutathione-S-transferase, luciferase, a polyhistidine tag, a T7 polymerase fragment and a secretion signal peptide.
  • Such derivatives may be prepared by fusing the peptides genetically or chemically.
  • the proteinaceous ICMMs or functional equivalents of the present invention may show some homology to Kazal type proteins.
  • the Kazal family of proteins includes a variety of protease inhibitors including pancreatic secretory trypsin inhibitor (Greene and Giordano, 1969), avian ovomucoid (Laskowski et al, 1987), acrosin inhibitor (Williamson et al, 1984) and elastase inhibitor (Tschesche et al. 1987).
  • the basic structure of a Kazal-type inhibitor is shown in the following schematic representation:
  • 'C conserved cysteine involved in a disulfide bond.
  • '#' active site residue.
  • ICMM shows homology with Kazal type proteins and believes that other Kazal type proteins may act as ICMMs.
  • a further embodiment of the invention therefore provides the use of a Kazal type protein, or functional equivalent thereof as an ICMM.
  • Functional equivalents of Kazal type proteins include fragments and variants of Kazal type proteins, providing that said fragments and variants retain ion channel modulatory activity.
  • the term functional equivalent is used to describe variants of Kazal proteins obtained by mutation or trunction of a Kazal type protein.
  • the ICMM comprises the amino acid sequence set out in Figure 9a, or a functional equivalent thereof. The ICMM comprising this sequence is referred to herein as EV048.
  • Functional equivalents of EV048 include variants, fragments, homologues or derivatives as defined above, providing that said variants, fragments homologues or derivatives retain activity as ion channel modulators.
  • Amino acids 1 to 20 of the EV048 amino acid sequence set out in Figure 9a form a signal sequence.
  • Functional equivalents of EV048 include fragments of the amino acid sequence set out in Figure 9a which do not contain the signal sequence.
  • variants, fragments, homologues or derivatives of EV048 should retain structural homology or conservation of the putative active site of EV048.
  • This active site may reside in the 7 amino acid sequence psggrrs between the third and fourth cysteine molecules in the sequence, although the Applicant does not wish to be bound by this theory.
  • a peptide or polypeptide comprising the sequence psggrrs as an ion channel modulator, as defined above.
  • the invention also provides a screening method for the identification of variants of EV048 which have enhanced ion modulatory properties compared to EV048.
  • variants of EN048, preferably mutants are created and these mutants are tested for the ability to modulate ion channel activity.
  • such mutants are tested for their ability to cause vasodilation and positive inotropism.
  • ICMMs or functional equivalents of the invention may be prepared in recombinant form by expression in a host cell. Suitable expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (2000) and Fernandez & Hoeffler (1998).
  • nucleic acid molecules comprising a nucleotide sequence encoding an ICMM or functional equivalent thereof, according to the first aspect of the invention.
  • nucleic acid molecules include single- or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acid species.
  • the nucleic acid molecules are DNA or cDNA molecules.
  • the invention also includes cloning and expression vectors incorporating the nucleic acid molecules of the second aspect of the invention.
  • Such expression vectors may additionally incorporate the appropriate transcriptional and translational control sequences, for example enhancer elements, promoter-operator regions, termination stop sequences, mRNA stability sequences, start and stop codons or ribosomal binding sites, linked in frame with the nucleic acid molecules of the second aspect of the invention.
  • appropriate transcriptional and translational control sequences for example enhancer elements, promoter-operator regions, termination stop sequences, mRNA stability sequences, start and stop codons or ribosomal binding sites, linked in frame with the nucleic acid molecules of the second aspect of the invention.
  • nucleic acid sequences encoding secretion, signalling and/or processing sequences may include nucleic acid sequences encoding secretion, signalling and/or processing sequences.
  • Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses), as well as other linear or circular DNA carriers, such as those employing transposable elements or homologous recombination technology. Many such vectors and expression systems are known and documented in the art (see, for example, Fernandez & Hoeffler, 1998). Particularly suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus- based vectors. Suitable hosts for recombinant expression include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities.
  • Mammalian cell lines grown in vitro axe also suitable, particularly when using virus-derived expression systems.
  • Another suitable expression system is the baculovirus expression system, that involves the use of insect cells as hosts.
  • An expression system may also constitute host cells that have the appropriate encoding nucleic acid molecules incorporated into their genome. Proteins, or protein fragments may also be expressed in vivo, for example in insect larvae or in mammalian tissues.
  • a variety of techniques may be used to introduce the vectors according to the present invention into prokaryotic or eukaryotic host cells. Suitable transformation or transfection techniques are well described in the literature (see, for example, Sambrook et al, 2000; Ausubel et al, 1991; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (e.g. episomal) or permanent (such as by chromosomal integration) according to the needs of the system.
  • the invention also includes transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule as defined above.
  • a further aspect of the invention provides a method for preparing an ICMM or a functional equivalent thereof as defined above, which comprises culturing a host cell containing a nucleic acid according to the invention under conditions whereby said protein is expressed and recovering said protein thus produced.
  • a further aspect of the invention provides a method for isolating an ICMM or functional equivalent thereof, as defined above comprising the steps of: preparing an extract from a haematophagous arthropod as defined previously; separating said extract into fractions containing different proteins; testing said fractions for the ability to modulate, preferably inhibit, ion channel activity; and isolating said ICMM or functional equivalent thereof, from a fraction(s) that possesses the ability to modulate, preferably inhibit, ion channel activity.
  • said extract is a salivary gland extract.
  • the preparation of such salivary gland extracts may take place at any one of several suitable points in the feeding cycle.
  • the extract is preferably prepared from the salivary glands of adult females since this is the only haematophagous instar.
  • an ICMM or functional equivalent thereof may be obtained by a method comprising the steps of: a) preparing a salivary gland extract from a haematophagous arthropod; b) separating said extract into fractions containing proteins; c) testing said fractions for the ability to modulate the activity of an ion channel; and d) isolating said ICMM or functional equivalent thereof from a fraction(s) that possesses the ability to modulate ion channel activity.
  • the haematophagous arthropod used in the above method is a horsefly of the Tabinadae family.
  • the ICMMs or functional equivalents are derived from horseflies of the Hybomitra, Heptatoma, Chrysops, Haematopota and Tabanus genera using the above-described method, more preferably, from Hybomitra bimaculata.
  • Suitable methods of separating haematophagous arthropod extracts into fractions containing purified proteins will be apparent to those skilled in the art.
  • the extract is separated into fractions of may be carried out using a chromatographic procedure, such as fast phase liquid chromatography (FPLC), high-performance liquid chromatography (HPLC), ion exchange chromatography, affinity chromatography, gel filtration or reverse phase HPLC.
  • FPLC fast phase liquid chromatography
  • HPLC high-performance liquid chromatography
  • ion exchange chromatography affinity chromatography
  • gel filtration or reverse phase HPLC reverse phase HPLC.
  • Testing of the fractions for their ability to modulate ion channel activity, in particular to cause vasodilation and/or positive inotropism and/or lengthen action potential can also be carried out by one of several methods known to those skilled in the art.
  • the methods used for assessing whether modulation of ion channels has been effected may vary depending on the ion channel under consideration. For example, in the case of the sodium-potassium ATPase, the activity of ATPase on varying concentrations of ATP in the presence and absence of fractions containing putative ICMMs may be assessed.
  • the effect of fractions on whole cell patch clamping in isolated cardiomyocytes or the effect on left ventricular output in an isolated perfused Langendorf rat heart may be assessed.
  • the effect on lengthening of action potential may be assessed by whole cell patch clamping in isolated cardiomyocytes.
  • Vasodilation may be assessed by the effect of the fractions containing putative ICMMs on pre-contracted rat femoral artery rings or by assessing the effect of fractions on coronary blood flow in an isolated Langendorf heart.
  • an ICMM or functional equivalent thereof may be isolated by any suitable procedures, including procedures such as SDS-polyacrylamide gel electrophoresis or two dimensional gel electrophoresis.
  • the present invention also includes an ICMM or functional equivalent thereof obtainable by any one of the methods described above.
  • the ICMM or functional equivalent thereof modulates the activity of a sodium channel, a potassium channel, a calcium channel and/or a sodium-potassium ATPase.
  • the ICMM or functional equivalent thereof may modulate the activity of more than one ion channel.
  • the ICMM or functional equivalent thereof exhibits the preferred ion channel modulatory properties listed above.
  • the method set out above may further comprise isolating and sequencing a gene encoding an ICMM, or functional equivalent thereof obtained using any of the methods set out above.
  • an isolated and purified ICMM may be subjected to a step of amino acid sequencing, followed by screening of a salivary gland gene library, for example using the polymerase chain reaction to isolate a gene encoding the ICMM.
  • a suitable procedure is by screening a cDNA library, optionally a cDNA expression library.
  • Certain expression libraries can be designed to generate tagged arthropod proteins, so facilitating their analysis and purification.
  • Suitable procedures for the preparation and isolation of parasite proteins can be found, for example, in co-owned patent applications PCT/GB97/01372 and PCT/GB98/03397.
  • a variety of suitable procedures for isolating a gene encoding an ICMM according to the invention will be known to the skilled reader.
  • a gene encoding an ICMM or functional equivalent thereof may be obtained by a method comprising performing the steps outlined in detail above to isolate the ICMM or functional equivalent thereof, and additionally performing the steps of: e) obtaining the N-terminal sequence of said isolated ICMM or functional equivalent thereof; f) designing a degenerate ohgonucleotide; and g) using said degenerate oligonucleotide to screen a salivary gland gene library to isolate a gene encoding the ICMM or functional equivalent thereof.
  • sequencing of the N- terminus of the protein may be carried out by any suitable method, as will be apparent to those skilled in the art.
  • a skilled person will readily be able to design one or more degenerate oligonucleotide probes or degenerate PCR primers which could encode this peptide sequence.
  • These primers may then be used to screen a salivary gland gene library, for example by hybridisation or by PCR.
  • a library is a cDNA gene library.
  • composition comprising an ICMM or functional equivalent, or a nucleic acid comprising a nucleotide sequence encoding an ICMM or a functional equivalent according to the invention in conjunction with a pharmaceutically acceptable carrier.
  • Pharmaceutically-acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.
  • Such carriers are well known to those of skill in the art.
  • such a composition may be used as a vaccine, and may thus optionally comprise an immunostimulating agent, for instance an adjuvant of the type referred to below.
  • an immunostimulating agent for instance an adjuvant of the type referred to below.
  • a process for the formulation of a vaccine composition comprising bringing an ICMM or functional equivalent, or a nucleic acid comprising a nucleotide sequence encoding an ICMM or a functional equivalent according to the invention into association with a pharmaceutically-acceptable carrier, optionally with an adjuvant.
  • Suitable adjuvants are well-known in the art and include oil-in-water emulsion formulations, saponin adjuvants, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IF A) and other substances that may act as immunostimulating agents to enhance the effectiveness of the vaccine composition.
  • CFA Complete Freund's Adjuvant
  • IF A Incomplete Freund's Adjuvant
  • the present invention provides for the use of an ICMM or functional equivalent thereof in therapy.
  • the invention also provides a method of treating an animal suffering from a disease or condition caused by a fault in ion channel activity, comprising administering to said animal an ICMM or functional equivalent thereof, a nucleic acid comprising a nucleotide sequence encoding an ICMM or a functional equivalent thereof or a pharmaceutical composition according to the invention in a therapeutically effective amount.
  • said animal is a mammal, more preferably a human.
  • conditions suitable for treatment using the ICMMs or functional equivalents of the invention include cardiac conditions such as coronary insufficiency leading to angina, congestive cardiac failure and cardiac arrhythmias; peripheral vascular disease such as cerebro-vascular insufficiency, intermittent claudication and Buerger's disease; vasospastic disorders such as Raynaud' s disease, cerebral or coronary vasospasm; reperfusion following stroke and myocardial infarction; shock including septic shock, haemorrhagic shock and cardiogenic shock; hypertension; to assist in circulatory support during and following cardio-pulmonary by-pass or angioplasty procedures.
  • cardiac conditions such as coronary insufficiency leading to angina, congestive cardiac failure and cardiac arrhythmias
  • peripheral vascular disease such as cerebro-vascular insufficiency, intermittent claudication and Buerger's disease
  • vasospastic disorders such as Raynaud' s disease, cerebral or coronary vasospasm
  • the invention also includes the use of an ICMM, variant or functional equivalent thereof as a diagnostic tool.
  • the invention includes the use of an ICMM, variant or functional equivalent thereof in the diagnosis of abnormalities of the cardiovascular system. Methods of diagnosis using an ICMM, variant or functional equivalent thereof will be well known to those skilled in the art.
  • the present invention also includes the use of an ICMM, variant or functional equivalent thereof, as a tool in the study of ion channel modulation and the effects of ion channel modulation, including vasodilation and inotropism.
  • Figure 1 Results of ⁇ PLC fractionation of crude Hybomitra bimaculata salivary gland extract.
  • SGE was applied to a Vydac C-4, 250 x 4.6 mm ID, 5 ⁇ m particle size column, UV monitored at 210 nm and 220 nm.
  • the active fraction from the first purification was further purified using a Beckman Ultrasphere C-18, 250 x 4.6mm ID, 5 ⁇ m particle size column and a gradient of 10-40% ACN with 0.1% TFA, flow rate 1 ml/min with 0.5% ACN/min increments, and monitored at 210 and 220 nm (Figure IB).
  • Figure IB For the third purification, a Vydac C 18, 250 x 4.6 mm ID, 5 ⁇ m particle size column was used under the same conditions as for the second purification (Figure IC).
  • Figure 2 In vitro effect of salivary gland extract (SGE) from Hybomitra bimaculata on the cardiac Na,K-ATPase activity. The SGE-quantity is expressed as amount of applied proteins from the extract.
  • SGE salivary gland extract
  • FIG. 3 In vitro effect of salivary gland extract (SGE) of Hybomitra bimaculata on kinetic parameters of Na,K-ATPase from rat heart.
  • SGE-quantity is expressed as amount of applied proteins from the extract.
  • the data represent means ⁇ SEM of 3 estimations, p ⁇ 0.05.
  • Figure 4 Vasodilating activity of Hybomitra bimaculata salivary gland extract on isolated rat femoral arterial rings with and without endothelium.
  • Figure 5 Vasodilating activity of protein HPLC fractions (retention time 10-28 minutes) of Hybomitra bimaculata salivary gland extracts on rat arterial rings without endothelium.
  • Figure 6 Effect of lOO ⁇ L Hybomitra bimaculata salivary gland extract on coronary blood flow in the isolated perfused rat heart.
  • Figure 7 Reverse phase HPLC of crude salivary gland extract of Hybomitra bimaculata. The peaks containing vasodilator activity are indicated with retention times.
  • Figure 8 N-terminal amino acid sequence of Hybomitra bimaculata salivary gland product EV048. Tentatively assigned residues after position 41 are indicated with a question mark.
  • Figure 9 Primary structure of Hybomitra bimaculata peptide EV048 (Figure 9a). Signal sequence underlined, cysteine residues shown in bold type, stop codon indicated by an asterisk. Alignment of peptide EV048 with Pfam consensus sequence for Kazal type proteins ( Figure 9b).
  • Figure 10 Pfam alignment of Hybomitra bimaculata peptide Ev049 with Kazal type proteins. Gaps in the alignment are indicated by dashes and dots. Residues in lower case are outstandingly different from the overall consensus.
  • THBI_RHOPR 6-48 and 57-101 Rhodnius prolixus thrombin inhibitor domain 1+2
  • IELA_ANESU/4-48 Anemonia sulcata inhibitor of elastase; AGRI, agrin; LAC, acrosin inhibitor; IOVO, ovomucoid inhibitor; QR1, quail retinal 1; SCI, secreted calcium binding 1 matric glycoprotein; SPRC, secreted protein acdic and rich in cysteine also called osteonedinand basement membrane protein 40 (bm40).
  • Figure 11 Coomassie blue stained NuPAGE 4-12% Bis-Tris gel showing purified peptide EV048. Lane (1) marker, (2-5) fractions h-k which elute at about 0.15M NaCl from a SP sepharose column. Marker sizes (kDa) indicated on left.
  • Figure 12 Effect of EV048 on isolated rat cardiomyocytes. Results of three experiments.
  • Figure 13 Effect of EV048 on coronary blood flow in isolated perfused rat heart.
  • Figures 13a and 13b show the results of two separate experiments.
  • Horsefly collection Horse flies were collected during the summer of 1999 in selected sites of south-western and western Slovakia using Manitoba traps. The effectiveness of the traps was improved by application of CO . Collections were performed during optimal weather conditions (sunny days, temperature 24 - 28°C, no wind) from May until the end of August. The collecting day started at 9:00 a.m. and finished at 5:00 p.m. Approximately 100-150 female horse flies per trap were collected each day of trapping, resulting in a total of 5,394 specimens. Horse flies were transported to the laboratory alive and then immediately processed to identify and isolate those of the species Hybomitra bimaculata.
  • the active fraction from the first purification was further purified using a Beckman Ultrasphere C-18, 250 x 4.6mm ID, 5 ⁇ m particle size column and a gradient of 10-40% ACN with 0.1% TFA, flow rate 1 ml/min with 0.5% ACN/min increments, and monitored at 210 and 220 nm ( Figure IB).
  • a Vydac C 18, 250 x 4.6 mm ID, 5 ⁇ m particle size column was used under the same conditions as for the second purification (Figure IC). Fractions were collected and dried in a Savant Instruments Speed- Vac. Protein sequence analysis
  • RNA/ terTM RNA/ terTM
  • cDNA was synthesised using a Stratagene cDNA synthesis kit (Cat # 200401-5). After fractionation into large and small cDNAs on a Sepharose CL-2B column, the ethanol precipitated cDNA pellets were each resuspended in 3.5 ⁇ l ddH O. cDNA yields were approximately 30ng/ ⁇ l and 200ng/ ⁇ l for the large and small molecules, respectively.
  • SM buffer 0.1M NaCl, 8mM MgSO 4 , 50mM TRIS.HC1 pH 7.5, 0.01% gelatin
  • Phage insert sizes were examined by PCR using T7 primers (5'TAA TAC GAC TCA CTA TAG 3') and T3 (5'AAT TAA CCC TCA CTA AAG 3').
  • Each lOO ⁇ l reaction comprised 2 ⁇ l eluted phage, 2 ⁇ l lOmM dNTPs, 2 ⁇ l of each primer (from stocks of 0.5 ⁇ g/ml), lO ⁇ l 10X REDTaq (Sigma) PCR reaction buffer (lOOmM Tris-HCl pH 8.3, 500mM KCl, l lmM MgCl 2 , 0.1% gelatin), 3 ⁇ l REDTaq (Sigma) DNA polymerase (1 unit/ ⁇ l in 20mM Tris-HCl, pH 8.0, lOOmM KCl, O.lmM EDTA, lmM DTT, 0.5% Tween 20, 0.5% Igepal CA-630, inert dye, 50% glycerol) and 79 ⁇ l ddH 2 O.
  • Thermal cycling (Hybaid Touchdown thermal cycler) parameters were IX 94°C 4min, 30X 94°C lmin, 48.5°C 45s, 72°C 90 s, and IX 72°C 5min. Agarose gel electrophoresis of the PCR products showed that large library inserts were >1600 base pairs and small library inserts ⁇ 1600 base pairs.
  • Cloning cDNA ofEV048 The N-terminal sequence determined for the HPLC fraction collected at 14.5 lmin (designated EV048) from H. bimaculata SGE was used to design three degenerate primers (HF1, HF2, HF3) for use with the T7 primer (which binds to the UniZAP XR vector), to amplify the cDNA encoding the peptide.
  • Each lOO ⁇ l reaction comprised 3 ⁇ l large or small library, 3 ⁇ l lOmM dNTPs, 2 ⁇ l T7 and 4 ⁇ l HF1 or HF2 or HF3 (from stocks of 0.5 ⁇ g/ml), lO ⁇ l 10X REDTaq PCR reaction buffer, 3 ⁇ l REDTaq DNA polymerase and 75 ⁇ l dH 2 O.
  • Thermal cycling parameters were IX 94°C 4min, 30X 94°C lmin, 48.5°C 45s, 72°C 90s, and IX 72 °C 5min.
  • Agarose gel electrophoresis revealed a range of PCR products. Five of these products were purified using a Qiaex II gel extraction kit (Qiagen) and sequenced with an ABI PRISMTM dye terminator cycle sequencing ready reaction kit and ABI sequencer (Perkin Elmer).
  • Qiaex II gel extraction kit Qiagen
  • ABI PRISMTM dye terminator cycle sequencing ready reaction kit ABI sequencer
  • a reverse primer (HR1 5'AAT ACA ACA TAT TCA AGT GG 3') matching the region beyond the stop codon was used with the T3 primer (which binds to the UniZAP XR vector) to obtain the 5' end of the cDNA.
  • the PCR product was cloned into the pGEM ® -T Easy vector (Promega) then sequenced revealing a full-length cDNA encoding EV048.
  • the HR1/T3 PCR product cloned into pGEM ® -T Easy was amplified using primer HF6 (5' GTA CGG ATC CAT GAA ATT TGC CTT GTT CAG T 3') which matches the signal sequence of EV048 and has a Bam HI restriction enzyme site (italic), and primer HR3 (5' CAT GCT GCA GTT AGT GAT GGT GAT GGT GAT GAC CCT TGC ACT CGC CAT CATG 3') which matches the sequence encoding the carboxy-terminal end of the protein and includes a codon for a glycine followed by six histidine residues (underlined) then a stop codon (bold) and Pstl restriction enzyme site (italic).
  • primer HF6 5' GTA CGG ATC CAT GAA ATT TGC CTT GTT CAG T 3'
  • primer HR3 5' CAT GCT GCA GTT AGT GAT GGT GAT GGT GAT GAC CCT
  • the lOO ⁇ l reaction comprised Ing HR1/T3 PCR product in pGEM ® -T Easy in a volume of l ⁇ l, 2.5 ⁇ l lOmM dNTPs, 2 ⁇ l HF6 and 2 ⁇ l HRl or HF2 (from stocks of 0.5 ⁇ g/ml), lO ⁇ l 10X REDTaq PCR reaction buffer, 3 ⁇ l REDTaq DNA polymerase and 77.5 ⁇ l dH 2 O.
  • Thermal cycling parameters were IX 94°C 4min, 20X 94°C lmin, 48.5°C 45s, 72°C 90s, and IX 72°C 5min.
  • the PCR product was gel purified, digested with Bam HI and Pst I and inserted into Bam HI and Pst I cut pFastBacl plasmid (Gibco-BRL®).
  • the sequence of the construct was verified by sequencing with primers PFBR (5' GAT TAT GAT CCT CTA GTA C 3') and PFBF (5' TAT TCC GGA TTA TTC ATA CC 3') which match pFastBacl either side of the multiple cloning site of the plasmid.
  • Transformation of the DHlO bacteria carrying the baculovirus DNA, purification of the baculovirus DNA and generation of high titre baculovirus stock were performed in accordance with the instructions accompanying the BAC-BAC ® baculovirus expression system (Gibco-BRL ® ).
  • S ⁇ cells grown in Sf-900 II serum free medium (Gibco-BRL ® ) to a cell density of 1 x 10 6 cells/ml were infected at a multiplicity of infection of 5 and grown for a further 60 h.
  • the cultures were centrifuged in a JA-20 rotor at 3000RPM for lOmin and the supernatant was poured into beakers kept on ice.
  • Polyethylene glycol MW 3350 was added to 30% (w/v) with stirring and the mixture was stirred for one hour.
  • the mixture was centrifuged in a JA-20 rotor at 5000RPM for 20min and the protein pellet was resuspended in 20ml binding buffer (50mM Na 2 HPO 4 /NaH 2 PO 4 pH 8, 500mM NaCl, 10% glycerol) per gram of wet paste.
  • 20ml binding buffer 50mM Na 2 HPO 4 /NaH 2 PO 4 pH 8, 500mM NaCl, 10% glycerol
  • the resin was then washed with 10 volumes lOmM TRIS .HCl pH 8 and then with 15 volumes of wash buffer (50mM Na 2 HPO 4 /NaH 2 PO 4 pH 6.5, 500mM NaCl, 10% glycerol). Bound proteins were eluted with 6 volumes lOOmM NaH 2 PO , 250mM imidazole and concentrated using Centricon 3 centrifugal filter devices (Amicon) spun in a JA-12 rotor at 5000RPM for 4 hours. Peptide EV048 was then purified further by cation exchange chromatography. The buffer flow rate was 1 ml/min for the washing and loading steps.
  • Cardiac sarcolemma was prepared from samples of the hearts of Wistar Kyoto rats by the hypotonic shock-Nal treatment method previously described (Vrbjar et al 1984). The protein content was assayed by the procedure of Lowry (Lowry et al 1951) using bovine serum albumin as a standard.
  • the substrate kinetics of Na,K- ATPase were estimated by measuring the splitting of ATP by 30-50 ⁇ g sarcolemmal proteins at 37°C in the presence of increasing concentrations of ATP in the range 0.08-4.0 mmol/1 in a total volume of 0.5 ml of medium containing 50 mmol/1 imidazole (pH 7.4), 4 mmol/1 MgCl 2 , 10 mmol/1 KCl and 100 mmol/1 NaCl. After 15 minutes of preincubation in the substrate free medium, the reaction was started by addition of ATP and 15 minutes later it was terminated by the addition of 1 ml of 12% solution of trichloroacetic acid.
  • SGE salivary gland extracts
  • Rat hearts were rapidly excised, placed in ice-cold perfusion buffer, cannulated via the aorta and perfused in the Langendorff mode at a constant perfusion pressure of 70 mm Hg and at 37°C.
  • Perfusion solution was a Krebs Henseleit buffer gassed with 95% O 2 and 5% CO 2 (pH 7.4) containing (in mM): NaCl 118.0, KCl 4.7, MgSO 4 1.66, CaCl 2 2.52, NaHCO 3 24.88, KH 2 PO 4 1.18 and glucose 5.55.
  • the solution was filtered through a 5 ⁇ m porosity filter (millipore).
  • An epicardial electrogram was registered by means of two stainless steel electrodes attached to the apex of the heart and the aortic cannula and continuously recorded (Miograph ELEMA-Siemens, Solna, Sweden). Heart rate was calculated from the EG. Coronary flow was measured by a timed (10 s interval) collection of coronary effluent which was weighed on an electronic balance (AND HF 200 G, A&D Company Limited).
  • Left ventricular pressure was measured by means of a latex water-filled balloon inserted into the left ventricle via the left atrium (adjusted to obtain end-diastolic pressure of 5-10 mm Hg) and connected to a pressure transducer (P23 Db Pressure Transducer, Gould Statham Instruments, Inc.)
  • Wistar rats 12 weeks old of both sexes were used. After they were killed humanely, two segments (each approx. 10 mm in length) of femoral artery were isolated and placed in a Krebs-Ringer bicarbonate solution comprising 118 mM NaCl, 5 mM KCl, 25 mM NaHCO 3 , 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 , 1 mM EDTA, 1.1 mM ascorbic acid, and 11 mM glucose. The endothelium was removed from one half of each segment preparation by gently rubbing the intimal surface.
  • the vessel segments were cleaned of adherent connective tissue and cut into 3 mm ring segments.
  • Two stainless-steel wires were passed through the lumen taking care not to damage the endothelium.
  • the rings were mounted on a myograph capable of measuring the isometric wall tension and placed within a bath containing Krebs-Ringer solution through which was bubbled 95% O 2 and 5% CO 2 , maintained at a temperature of 37°C and pH 7.4.
  • the effectiveness of endothelium removal was demonstrated by failure of acetylcholine (5x10 -6 mol/1) to relax a contraction induced by phenylephrine (5xl0 "6 mol/1).
  • the plateau of the contractile response induced by phenylephrine (5x10 ⁇ 6 mol/1) was taken as a measure of 100% contraction.
  • Rat ventricular myocytes were isolated by enzymatic digestion, layered in a glass perfusion chamber mounted on the stage of an inverted microscope, and perfused with a salt solution containing (mM): NaCI 140, KCl 54, CaCI 2 1.0, MgCl 1.0, glucose 10.0 and HEPES 10.0 (pH 7.35 adjusted with NaOH).
  • the cells were field stimulated to contract at 0.5 Hz with 1.0 ms square wave pulses of supra-threshold voltage. The decrease in cell length during each contraction was measured with an edge detection system using a photodiode array. The temperature of the superfusate was 24 ⁇ 2°C. Data were digitised and acquired on a personal computer via an A/D converter using VCLAMP software (CED, Cambridge, UK).
  • an Axoclamp 200 amplifier (Axon Instruments, Inc., Burlingame, Calif., USA) was used in whole-cell voltage clamp or current clamp mode to record ionic currents and action potentials.
  • Patch pipettes were pulled from filamented borosilicate capillary glass (GC150TF; Clark Electromedical Instruments, UK) on a microprocessor-based three-stage puller (Mecanex BB-CH-PC, Basel, Switzerland). The pipettes had resistances of 2-4 M ⁇ after filling with internal solution.
  • the pipettes were filled with a solution containing (in mM): KCl 140, MgCl 2 1.0, Mg-ATP 5.0, Na 2 -phosphocreatine 5.0, HEPES 5.0 (pH 7.2 adjusted with KOH).
  • the external solution for these experiments was (mM): NaCl 140, KCl 5.4, CaCl 2 1.8, MgCl 2 1.0, glucose 10.0 and HEPES 5.0 (pH 7.4 adjusted with NaOH).
  • Action potentials were elicited by injection of short current pulses.
  • Ic a was elicited by applying 100 ms depolarising voltage pulses in 5 mV steps from a holding potential of -45 mV.
  • the pipette was filled with a solution containing (mM): KCl 145, MgCl 2 3.0, Mg-ATP 5.0, Na 2 -phosphocreatine 5.0, EGTA 5.0 and HEPES 5.0 (pH 7.2 adjusted with KOH).
  • SGE from Hybomitra bimaculata contains at least one compound which at lower concentrations stimulates Na,K- ATPase but at the highest concentration tested (6.5 ⁇ g) has an inhibitory effect.
  • This biphasic reaction suggests that at lower concentrations the salivary gland extract is able to increase the hyperpolarisation of muscle cells but at higher concentrations it has the reverse effect.
  • Table 1 Effect of salivary gland extract from Hybomitra bimaculata on the isolated perfused rat heart.
  • FIG. 7 demonstrates the RP-HPLC chromatogram obtained from SGE of 475 pairs of salivary glands. A vasorelaxation activity of 47% was found in the peak with a retention time of 13.77 min; 45% relaxation was measured in the peak with a retention time of 16.28 min. Less activity (30%) was obtained with a peak of retention time 9.51 min, and 15% with a peak of retention time 22.47 min. Amino acid analysis and sequencing of Hybomitra bimaculata HPLC fractions
  • the full length cDNA for EV048 encodes a peptide of 76 amino acids ( Figure 9a). This includes a 20 amino acid putative signal peptide that is probably cleaved at VAA - DEC to generate the mature N-terminus ( Figure 8).
  • the complete peptide has a predicted molecular weight of 8282.4 Da and a theoretical pi of 8.27.
  • the 56 amino acid mature peptide has a predicted molecular weight of 6146.7Da and a theoretical pi of 7.78.
  • An N- linked glycosylation site is predicted at the asparagine residue at position 26 in the mature peptide; there are no predicted O-linked sites.
  • the sequence of the mature peptide has similarity with Kazal-type protease inhibitors (Figure 10), including homology with rhodniin I and II, the Kazal-type inhibitors from Rhodnius prolixus, another haematophagous insect species (Friedrich et al., 1993, van de Locht et al., 1995), and with the protease inhibitor from the sea anemone Anemonia sulcata that is specific for elastases (Tschesche et al., 1987).
  • 'C conserved cysteine involved in a disulfide bond.
  • '#' active site residue.
  • Homology modelling indicates that the insertion occurs within, or just before, the region of the peptide that forms the second ⁇ -sheet.
  • the highly conserved tyrosine residue (shown in bold in Figure 10) is a phenylalanine in EV048, and the putative active site residue (indicated by # in bold in Figure 10) is an alanine.
  • the expressed peptide is exported from the cell to the supernatant.
  • Expression levels of EV048 in the supernatant were approximately 0.3 ⁇ g per ml of Sf9 cells.
  • the expressed protein is approximately the size (7 kD) expected for the mature peptide (with glycine and 6 X HIS tag) and was purified to homogeneity (Figure 11) in two steps.
  • Tachykinins elicit release of nitric oxide following binding of the peptide to endothelial cell tachykinin receptors. Such binding induces endothelium-dependent vasorelaxation (Champagne and Ribeiro, 1994).
  • protein vasodilators such as nitrophorins are able to bind and release nitric oxide. Delivering NO to the host vessel induces direct relaxation of smooth muscle by increasing intracellular cGMP levels (Champagne, 1994; Weichsel et al, 1998).
  • Other vasodilators like maxadilan increase the intracellular level of cAMP within smooth muscle cells leading to relaxation (Grevelink et al., 1995).
  • vasodilating mechanisms are endothelium-independent. It is proposed that, unlike vasodilators found in other haematohagous arthropod species, EV048 does not act either as a tachykinin or as a NO donor.
  • the SGE from Hybomitra bimaculata contains at least one compound which, at low concentration, stimulates Na,K-ATPase but at higher concentration inhibits the enzyme.
  • 3 ⁇ g of SGE were applied it induced a significant stimulation of the Na,K-ATPase.
  • Increasing the amount of SGE to 6.5 ⁇ g induced an inhibition of the Na,K-ATPase.
  • This phenomenon may be fundamental to understanding the vasodilating activity of this SGE on the rat femoral artery, whilst also explaining the positive inotropism of the SGE from Hybomitra bimaculata in the isolated rat heart and of the recombinant molecule EV048 in isolated rat cardiomyocytes.
  • the duration of action after removal of the active agent by washout (> 16 minutes) is also considered to increase its potential value as a therapeutic agent. Prolongation of the action potential without apparently inducing spontaneous oscillations may also make it a useful anti-arrhythmic agent.
  • Kazal-type protein from Hybomitra bimaculata is also considered to increase its potential value as a therapeutic agent. Prolongation of the action potential without apparently inducing spontaneous oscillations may also make it a useful anti-arrhythmic agent.
  • the Kazal family of proteins includes a variety of protease inhibitors including pancreatic secretory trypsin inhibitor (Greene and Giordano, 1969), avian ovomucoid (Laskowski et al, 1987), acrosin inhibitor (Williamson et al, 1984) and elastase inhibitor (Tschesche et al. 1987).
  • Kazal inhibitors contain between 1 and 9 Kazal-type inhibitor repeats.
  • Kazal protease inhibitors that inhibit trypsin-like proteinases have basic residues (R, K or H) at their active (or PI) site whereas those that inhibit chymotrypsin-like proteases have large hydrophobic residues at the PI position.
  • the putative active site residue of EV048 is the small hydrophobic amino acid alanine. This residue is not present in any other Kazal proteins known to inhibit proteases. However, the active site of EV048 appears similar to a modelled sequence predicted to have very tight binding to porcine pancreatic elastase (Lu et al, 2001).
  • the extra sequence (PSGGRRS) inserted between the third and fourth cysteine residues of EV048 may well play a role in the vasodilating properties of the peptide. Homology modelling suggests that the additional amino acids exist at an exposed location and may permit interaction with a target molecule. Initial studies with the recombinant molecule presented here have demonstrated biological activity consistent with the effects observed with crude SGE from Hybomitra bimaculata. References
  • Lu SM, Lu W, Qasim MA, Anderson S, may be treated with Ardelt W, Bigler T, Chiang YW, Cook J, James MN, Kato I, Kelly C, Kohr W, Komiyama T, Lin TY, Ogawa M, Otlewski J, Park SJ, Qasim S, Ranjbar M, Tashiro M, Warne N, Whatley H, Wieczorek A, Wieczorek M, Wilusz T, Wynn R, Zhang W, Laskowski M Jr (2001). Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases. Proc Natl Acad Sci USA, 98, 1410-5

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Abstract

La présente invention concerne des modulateurs de canaux ioniques, notamment des modulateurs de canaux ioniques dérivés d'arthropodes hématophages. L'invention concerne également l'utilisation de modulateurs de canaux ioniques d'arthropodes hématophages dans le traitement et la prévention de certaines maladies et de certains états chez les mammifères, notamment les êtres humains.
EP02748981A 2001-06-22 2002-06-21 Modulateurs de canaux ioniques Withdrawn EP1401868A2 (fr)

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