EP1054896A1 - PEPTIDES ANALGESIQUES EXTRAITS DU VENIN DE LA $i(GRAMMOSTOLA SPATULATA) ET LEUR UTILISATION - Google Patents

PEPTIDES ANALGESIQUES EXTRAITS DU VENIN DE LA $i(GRAMMOSTOLA SPATULATA) ET LEUR UTILISATION

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Publication number
EP1054896A1
EP1054896A1 EP98905534A EP98905534A EP1054896A1 EP 1054896 A1 EP1054896 A1 EP 1054896A1 EP 98905534 A EP98905534 A EP 98905534A EP 98905534 A EP98905534 A EP 98905534A EP 1054896 A1 EP1054896 A1 EP 1054896A1
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European Patent Office
Prior art keywords
cys
lys
glu
leu
arg
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EP98905534A
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German (de)
English (en)
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Richard Alexander Lampe
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AstraZeneca AB
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Zeneca Ltd
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    • 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
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides that induce analgesia in mammals. More particularly, the present invention relates to analgesia-inducing peptides obtainable from venom of Grammostola spatulata, the Chilean pink tarantula spider. BACKGROUND OF THE INVENTION
  • Pain is one of the basic clinical symptoms seen by every physician and is usually categorized into three segments: mild, moderate and severe.
  • the mild-to- moderate segment has multiple product entries including aspirin, acetaminophen, ibuprofen, and other non-steroidal, anti-inflammatory (NSAID) products.
  • Narcotic analgesics remain the mainstay of currently marketed products for the treatment of moderate-to-severe pain.
  • Cancer and the post-operative surgical period are two conditions most often associated with moderate-to-severe pain.
  • Tumor infiltration of bone, nerve, soft tissue or viscera are the most common causes of cancer pain accounting for 65-75% of patients. Pain as a result of cancer treatment from surgery, chemotherapy or radiation accounts for 15-25% of patients, with the remaining 5-10% reporting pain independent of their cancer or cancer therapy.
  • Various factors influence the prevalence of cancer pain including the primary tumor type, stage and site of disease and patient variables, especially psychological variables.
  • patient response to post surgical pain is dependent upon location and extent of intervention as well as personal attributes.
  • post surgical pain is distinguished from cancer pain by length of treatment period.
  • narcotics which constitute the largest segment of the U.S. market for treatment of moderate-to-severe pain, is the potential for addiction and loss of activity (i.e. tolerance) with continued use. Consequently, there is a need for other analgesics that can relieve pain, especially moderate-to-severe pain associated with caner.
  • newer drug delivery strategies include transdermal narcotics, PCA, intraspinal implantation of controlled release pumps and implantation of encapsulated living cells which release naturally-occurring endorphins or other analgesic peptides.
  • New drug approaches reflect the varying pathways and causes of moderate-to severe pain.
  • Classes of compounds in development for treating pain include serotonergics, noradrenergics, opioid partial agonists and kappa opioid agonists.
  • Therapeutic targets with significant preclinical investigation include tachykinin/bradykinin antagonists and excitatory amino acid antagonists. Newer targets being exploited include growth factors, cytokines, nitride oxide regulators, etc. Natural sources including folk medicine remedies and frog venom extracts are also under investigation.
  • GsAF I Cys-Lys-Lys-Arg-Leu-NH2 (referred to herein as GsAF I) (SEQ ID NO: 1) 3 or
  • GsAF LI Cys-Lys-Lys-Lys-Ile-Glu-Trp
  • the present invention thus provides for the use in the manufacture of a medicament for treatment of pain of the peptides of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention further provides for the use of the peptides of SEQ LD NO: 1 or SEQ ID NO: 2 in the treatment of pain.
  • An additional aspect of the invention provides a purified peptide having the amino acid sequence
  • compositions comprising a pharmaceutically acceptable carrier or diluent and a peptide having the amino acid sequence Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
  • Yet another aspect of the invention provides methods for identifying compounds that mimic the analgesia-inducing activity of GsAF I and/or GsAF II.
  • the present invention additionally provides antibodies specific for GsAF I.
  • the antibodies can be monoclonal or polyclonal.
  • Antibodies can be prepared using methods known in the art such as the methods in Harlow et al. eds., Antibodies: A Laboratory Manual, New York, cold Spring Harbor Laboratory Press (1988). 4 DETAILED DESCRIPTION OF THE INVENTION
  • peptides from venom of the Chilean pink tarantula spider, Grammostola spatulata have analgesia-inducing properties and are thus useful as analgesics for treatment of pain in mammals, including humans, and as research tools for identification of compounds that mimic the analgesic activity of the peptides.
  • the present invention provides a method for treating pain comprising administering to a mammal in need of such treatment an effective analgesic amount of a peptide having the amino acid sequence Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys- Asp-Ser-Glu-
  • the peptides are useful for treating pain in mammals, including humans, conventional laboratory animals such as rats, mice and guinea pigs, domestic animals such as cats, dogs and horses, and any other species of mammal.
  • the peptides can be used to treat acute or chronic pain from any source or condition, such as burns, cancer, neuropathies, organ inflammation or surgical intervention. Preferably, however, the peptides are used to treat moderate-to-severe pain due to cancer or surgery.
  • the peptides can be administered orally, parenterally, intrathecally, topically, intraveneously, intramuscularly or intradermally/epineurally. A preferred route of administration is intrathecally.
  • the peptides thereof can be prepared for pharmaceutical use by inco ⁇ orating them with a pharmaceutically acceptable carrier or diluent.
  • a further aspect of the present invention provides pharmaceutical compositions comprising a peptide from Grammostola spatulata as described herein and a pharmaceutically acceptable carrier or diluent.
  • the peptide can be prepared for pharmaceutical use by inco ⁇ orating it in unit dosage form as tablets or capsules for 5 oral or parenteral administration either alone or in combination with suitable carriers such as calcium carbonate, starch, lactose, talc, magnesium stearate, and gum acacia.
  • the peptide can be formulated for oral, parenteral or topical administration in aqueous solutions, aqueous alcohol, glycol or oil solutions or oil-water emulsions. Buffered- aqueous or carrier mediated aqueous/non-aqueous intrathecal and intraveneous dosages can be formulated. These and other suitable forms for the pharmaceutical compositions of the invention can be found, for example, in Remington's
  • compositions of the invention can comprise any combination of one or both of the peptides.
  • the amount of the active component (i.e. peptide) in the pharmaceutical compositions can be varied so that a suitable dose is obtained and an effective analgesic amount can be administered to the patient.
  • the dosage administered to a particular patient will depend on a number of factors such as the route of administration, the duration of treatment, the size and physical condition of the patient, the potency of the peptide and the patient's response thereto.
  • An effective analgesic amount of the peptide when administered intrathecally is generally in the range of from about 5 nanograms per kilogram body weight of the patient to about 500 micrograms per kilogram; preferably from about 50 nanograms per kilogram to about 50 micrograms per kilogram; more preferably from about 500 nanograms per kilogram to about 5 micrograms per kilogram.
  • Effective amounts of the peptide will vary when administered by other routes.
  • An effective analgesic amount can be estimated by testing the peptide in one or more of the pain tests disclosed herein to arrive at a dose that can be varied according to one or more of the criteria listed above to provide a suitable amount of the peptide to the mammal.
  • inducing analgesia refers to the ability of the peptide to treat pain in mammals or attenuate pain as evidenced by favorable results in one or more conventional laboratory models for testing pain or assessing analgesia such as the tests set forth herein.
  • Analgesic activity of the peptides is determined by testing in at least one, and preferably more than one, of a series of tests which includes 1) tail flick latency 6 (Abbott, F.V. etal, Pharmacol. Biochem. Behav., 17, 1213-1219, 1982; Cridland,
  • the tail flick latency and hot plate threshold tests are measurements of thermal nociception.
  • the von Frey filament threshold test evaluates mechanical nociceptive activity. All three of these pain tests evaluate the analgesic activity of compounds against the phasic stimulation of either thermal- or mechanical- nociceptors and reflect to a large degree the activation of A- and poly modal C-fiber afferents.
  • Clinical analgesics with an opioid-based mechanism of activity are efficacious in these tests, whereas those analgesics which either interact preferentially with peripheral targets or possess multiple sites of action are generally less active. These tests are good predictors of moderate to strong analgesic agents and within the opioid class of compounds the correlation with clinical effect is good.
  • the non- steroidal anti-inflammatory (NSAID) class of analgesics which clinically target the lower end of the pain scale, are not routinely detected under the parameters normally used for these tests.
  • Analgesic detection of NS AIDs is dependent upon the generation of a nociceptive status of increased responsiveness (i.e. a lowering of threshold to noxious stimuli) in response to primary afferent tissue damage and inflammation. Interaction between the immune and nervous systems to induce this state represents the target for NSAID activity. Inhibition of this heightened activity of peripheral nociceptors, and of the corresponding central circuitry, is detected over longer time intervals by either monitoring spontaneous behavior or the response to subsequent noxious stimuli. These more chronic measurements of the "hyperalgesic" status are considered to mimic most clinical conditions of pain. They also broaden the detection capability for useful analgesic agents without exclusion of active agents detected in the phasic pain tests.
  • the noxious stimuli used to induce this condition are either chemical irritants/caustic agents or inflammatory stimulators.
  • the major defining variable is the time interval required for the development, and the ethically 7 justifiable duration, of the hyperalgesic/inflammatory state.
  • Compounds can be evaluated for their intrinsic activity to prevent the development of the hyperalgesic condition (i.e. compound administered prior to noxious stimulant) or to reduce the increased nociceptive response (i.e. compound administered post-noxious stimulation) or both.
  • Primary end points in these tests are measurements of nociceptive and inflammatory status.
  • the peptides are useful in biological assays such as assays to detect compounds that mimic the analgesic activity of the peptides, assays to detect the anatomical site of action of the peptides, or studies on the mechanism of action of the peptides.
  • Another aspect of the invention provides methods for detecting compounds that mimic the analgesic activity of GsAF I and/or GsAF II. Mimicking the activity of the peptides disclosed herein refers to the ability of test compounds to induce analgesia, bind to cellular receptors to which the peptides bind or otherwise act in the same or similar physiological manner as the peptides.
  • the present invention provides methods for identifying compounds having analgesia-inducing activity or which otherwise mimic the activity of GsAF I and/or GsAF II comprising the steps of adding a test compound to a biological assay that determines activity of a peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting the activity of the test compound.
  • Biological assays to identify compounds that mimic the activity of GsAF I and/or II can be in vivo assays, such as those described herein, or in vitro assays, such as the assays described below.
  • GsAF I and/or GsAF II can be used in competitive binding screening assays to identify compounds that mimic the activity of GsAF I and II according to the following method.
  • a test compound and detectably labeled peptide are added to mammalian cells or tissue under conditions that allow binding to the cells or tissue. Binding of labeled peptide to the mammalian cells or tissue is then measured. Compounds that mimic the activity of the detectably labeled peptide will compete with the peptide for binding sites on the receptor.
  • GsAF I and/or II could be labeled with 1 ⁇ 1 anc i U sed in the assay described in Stumpo et al, Eur. J. Pharmacol. 206:155, 1991 and modified from Abe et al, Neurosci. Lett. 71:203, 1986.
  • test compounds are preincubated with brain or spinal cord membrane tissue prior to the addition of ⁇ I-labeled GsAF I and/or II, followed by incubation to allow binding to occur.
  • the reaction mixture is then filtered and the filters containing the brain or spinal cord membrane tissue are rinsed with buffer.
  • Binding of 125j_ labeled peptide can be determined by scintillation counting.
  • Compounds that mimic the action of GsAF I and II will compete with the labeled peptide and produce lower levels of labeled peptide binding to the receptor on the cells of the brain or spinal cord membrane tissue than compounds that do not mimic the activity of GsAF I or LI.
  • Nonspecific binding will be defined as that remaining in the presence of excess (100- 1 ,000X) unlabeled GsAF I or GsAF II.
  • the peptides preferably inco ⁇ orate a detectable label.
  • the detectable label can be any conventional type of label and is selected in accordance with the type of assay to be performed.
  • the detectable label can comprise a radiolabel such as ⁇ C, ⁇ l, or ⁇ H, an enzyme such as peroxidase, alkaline or acid phosphatase, a fluorescent label such as fluoroisothiocyanate (FTTC) or rhodamine, an antibody, an antigen, a small molecule such as biotin, a paramagnetic ion, a latex particle, an electron dense particle such as ferritin or a light scattering particle such as colloidal gold.
  • FTTC fluoroisothiocyanate
  • Suitable method to detect such labels include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement or light emission measurement.
  • Detectable labels procedures for accomplishing such labeling and detection of the labels are well known in the art and can be found, for example, in An Introduction to Radioimmunoassays and Related Techniques: Laboratory Techniques in Biochemistry and Molecular Biology, 4th Ed., T. Chard, Elsevier Science Publishers, Amsterdam, The Netherlands, 1990; Methods in Non-Radioactive Detection, Gary C. Howard, Ed., Appleton and Lange, East Norwalk, Ct, 1993 or Radioisotopes in Biology: A Practical 10 Approach. R.J. Slater, Ed., IRL Press at Oxford University Press, Oxford, England,
  • the peptides can be used in the assay of Keith et al, J. Auton. Pharmacol., 9:243-252, 1989 and Mangano et al, Eur. J. Pharmacol. 192:9-17, 1991 to identify compounds that mimic the activity of GsAF I or LI. Briefly, this assay measures K + - evoked release of ⁇ H-D-aspartate and - > H-norepinephrine from rat brain or spinal cord slices. Spinal cord or brain slices can be pre-equiHbrated with GsAF I/GsAF II, test compound or vehicle for 15 min prior to K + stimulation.
  • test compounds used in the screening assay are small organic molecules but analgesic activity of any type or size of compound such as proteins and peptides can also be tested with the methods of the invention.
  • GsAF I and/or II can be used in assays to identify its site of action and for further physiological characterization of its activity.
  • the peptides can be used to study inhibition of binding/interaction of labelled ligand to mammalian tissues, isolated cells or subcellular components derived therefrom.
  • the peptides can be used to study inhibition of binding/interaction of labelled ligand to specific recombinantly expressed proteins generated following either cDNA or genomic transformations/transfections of eukaryotic or prokaryotic host systems.
  • the peptides can be used to study analogous biochemical interaction with mammalian tissue function to include receptor mediated activation/inhibition of specified transduction pathways, movement of ionic species across biological membranes and alteration of transcriptional/translational profile of specific pain-induced gene activity.
  • 11 methods to measure the alteration of potassium, sodium, calcium, chloride or hydrogen ionic distribution across mammalian cell derived membrane barriers as measured by either radioisotopic or fluorescent detection of specified ionic species can be utilized. The effects of these ionic movements upon the regulation of specific immediate early genes can be studied as well.
  • the peptides can additionally be used for electrophysiological measurements of potassium, sodium, calcium and chloride distribution across mammalian cell membranes to include macroscopic analysis of synaptic transmission as well as microscopic analysis of specified ionic currents. Specifically, inhibition of noxious-mediated neuronal firing and synaptic transmission within spinal dorsal horn neurons can be analyzed as well as inhibition of isolated specific ionic currents within individual dorsal root ganglion or spinal dorsal horn neurons.
  • the peptides can further be used in studies of inhibition of physiological response to nociofensive/noxious stimuli administered to mammalian species. Specifically, motor parameters (i.e. limb withdrawal thresholds or response time latencies/durations) can be quantitated in response to either thermal, mechanical or chemical noxious stimuli administered to either naive animals or animals in which a painful condition has been experimentally induced.
  • motor parameters i.e. limb withdrawal thresholds or response time latencies/durations
  • GsAF I and II can be prepared by isolation from Grammostola spatulata venom, chemical synthesis or recombinant DNA methods.
  • Grammostola spatulata venom is commercially available from Spider Pharm, Feasterville, Pennsylvania, USA.
  • the peptides are preferably isolated from spider venom by sequential fractionation using reverse phase-high pressure liquid chromatography on C-8 and C- 18 silica supports with trifluoroacetic acid/acetonitrile buffer.
  • a preferred C-8 silica support is Zorbax® Rx C-8 (Mac-Mod Analytical, Inc., West Chester, Pennsylvania) which is comprised of 5 micron diameter silica particles having 300 A pore size and covalently modified to contain diisopropyloctyl side chains.
  • the C-18 silica support is preferably comprised of 5 micron diameter silica particles having 30 ⁇ A pore size and covalently modified to contain an octadecyl side chain.
  • Other types of C-8 and C- 18 silica supports are also suitable for use in isolating the peptides.
  • a preferred buffer is 0.1% trifluoroacetic acid in acetonitrile.
  • crude venom is initially fractionated on a C-8 semi-preparative column using a broad 20-50% 12 gradient of 0.1% trifluoroacetic acid in acetonitrile buffer.
  • the peptides are further purified using a C-8 column and shallower gradients of the same buffer, followed by additional fractionation using a C-8 column and the broad buffer gradient.
  • GsAF I and II can be prepared by recombinant DNA techniques.
  • a DNA sequence coding for one of the peptides is prepared, inserted into an expression vector and expressed in an appropriate host cell.
  • the peptide thus produced is then purified from the host cells and/or cell culture medium.
  • Methods for preparing DNA coding for the peptides and expression of the DNA are well-known and can be found, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, Guide to
  • peptides can also be prepared by chemical synthesis using either automated or manual solid phase synthetic technologies. These techniques are well known in the art and are differentiated on the basis of features such as selection of synthetic resin backbone, selection of amino, carboxyl and side chain protecting groups and selection of deprotection strategies. Methods for synthesizing peptides can be found in standard texts such as E. Atherton and R.C. Sheppard, Solid Phase
  • synthesis of GsAF I and GsAF II can be done using Fmoc chemistry on an automated synthesizer.
  • production of the linear reduced peptide can be performed in either a single process or in two different processes followed by a condensation reaction to join the fragments.
  • a variety of protecting groups can be inco ⁇ orated into the synthesis of linear peptide to facilitate isolation, purification, and/or yield of the desired peptide. Protection of cysteine residues found in the peptide can be accomplished using, for example, a triphenylmethyl, acetamidomethyl and/or 4- methoxybenzyl group in any combination. Such a strategy may offer advantages for 13 subsequent oxidation studies to yield folded peptide.
  • a further aspect of the invention provides a novel peptide which has the amino acid sequence
  • the leucine at the carboxy terminus of the peptide is amidated, i.e., the free end of the terminal leucine residue ends with -CO-NH2 instead of -COOH. Both amidated and non-amidated peptides are within the scope of the present invention.
  • a purified or isolated peptide refers to a peptide that is substantially free of contaminating cellular components, other venom constituents or other material such as reagents used in chemical synthesis of the peptide.
  • the peptide is present in a mixture containing the peptide in an amount greater than about 50% of the total mixture, more preferably in an amount greater than about 80%, most preferably in an amount greater than about 90%.
  • Example 1 Isolation and Characterization of Peptide GsAF I from Venom of Grammostola spatulata A. Isolation of Peptide Crude Grammostola spatulata venom was supplied as frozen aliquots by
  • Zorbax® RX-C8 semipreparative column was done with a 20-50% gradient of TFA/CH3CN Buffer (0.1% trifluoroacetic acid in acetonitrile) over 30 minutes with a
  • Fraction 18 (and optionally 17) were applied to a Zorbax ® RX-C8 semipreparative column and fractionated using a 24-30% gradient of TFA/CH3CN Buffer over 24 minutes, with 3 minute delay.
  • the major UV absorbing peak was manually 15 collected with removal of peak tails. After this step, sample purity was usually found to be at least 85%.
  • the major UV absorbing peak collected in the previous step was further purified using a 20 - 50% gradient of TFA/CH3CN Buffer on a Zorbax ® RX-C8 semi-preparative column (flow rate 5 ml/min) over 30 min with a 3 minute delay.
  • the primary peak which elutes at 22 minutes was collected manually with removal of peak tails.
  • GsAF I sample purity was found to be about 98% pure.
  • Flow injections containing approximately 200-400 picomoles of peptide were measured.
  • the average molecular weight of GsAF I was determined to be 3707.5 Daltons (Da). After thiol reduction, the average molecular weight was measured at 3713.5 Daltons. Since each reduction of a disulfide bond increases the mass of a peptide by 2 Da, the peptides contain three disulfide linkages based upon the 6 Da mass shift.
  • the native oxidized peptide was digested with a combination of modified trypsin (Boehringer Mannheim) and endoproteinase Asp-N proteases. The resulting mixture of proteolysis products was subjected to liquid chromatography-electrospray mass spectral analysis to assign disulfide linkage. Multiple peptides containing a 16 disulfide bridge linking amino acids 9 and 21 of the GsAF I peptide were observed.
  • N-terminal sequencing was performed on a gas phase sequencer (Applied Biosystems 475, Foster City, CA). SDS-Page was performed using a 16.5% high cross linked Tris-Tricine gel (Schagger,H. and G. von Jagow, Anal. Biochem. 166:368-379, 1987) and electroblotted to ProBlot (Applied Biosystems, Foster City, CA)) as described by Matsuidara, P., J. Biol. Chem. 262:10035-10038. Electroblotted bands were pyridylethylated in the gas phase according to the method described in Andrews, P.C. and J.E. Dixon, Anal. Biochem.
  • Example 2 Isolation and Characterization of Peptide GsAF II A. Isolation of Peptide Crude Grammostola spatulata venom was supplied, as frozen aliquots, by the commercial vendor Spider Pharm, Inc. (Feasterville, Pennsylvania 19053, USA). Reverse phase-high pressure liquid chromatography (RP-HPLC) of the venom was performed using Zorbax® Rx-C8 semi-preparative (25 cm x 9.4 mm) and analytical (25 cm x 4.6 mm) columns (Mac-Mod Analytical, Inc.
  • RP-HPLC Reverse phase-high pressure liquid chromatography
  • Zorbax® Rx-C8 is comprised of 5 micron silica microsphere particles having a 30 ⁇ A pore size and covalently modified with diisopropyl octyl side chains) and a C-18 analytical (25 cm x 4.6 mm) column (Vydac, Hesperia, CA; the C-18 support is comprised of 5 micron silica microsphere particles having a 300A pore size and covalently modified with octadecyl side chains).
  • Semi-preparative scale RP-HPLC was done using a 5 milliliter/minute flow rate whereas a 1 milliliter per minute flow rate was used for the analytical analyses. 18
  • Detection of eluting entities were monitored via ultraviolet (UN) spectroscopy at 215 nm and fractions were either collected at 1 minute intervals or manually based upon UN intensity.
  • Initial injection volumes of 30-50 microliter ( ⁇ l) crude venom were made. Consequently, multiple fractionations were carried out at each stage of the purification with pooling of individually identical fractions. All fractions were lyophilized prior to resuspension in HPLC grade H2O for subsequent purification or in vitro testing. Resuspension volumes were based upon original crude venom volumes. Evaluation was done on samples deemed to be greater than 90% homogeneous by RP-HPLC. Samples were stored at 4 C following resuspension. No detectable loss of activity was witnessed with storage or with adherence to either plastic or glass.
  • Fraction 19 was applied to a Zorbax ® RX-C8 semi-preparative column and fractionated using either a 29-33% or a 30-34% gradient of TFA/CH3CN Buffer over 24 minutes with a 3 minute delay.
  • the major UV absorbing peak was manually collected with removal of peak tails. After this step, sample purity was usually found to be at least 85%.
  • the major UV absorbing peak was further purified using a 20- 50% gradient of TFA/CH3CN Buffer over 30 min with a 3 minute delay. The primary peak which elutes at 23.5 minutes was collected manually with removal of peak tails. GsAF II sample purity was found to be about 98% pure.
  • the peptide GsAF II was characterized using the methods described for peptide GsAF I in Example 1.
  • the average molecular weight of GsAF II was determined to be 3979.9 Daltons (Da). After thiol reduction, the average molecular 19 weight was 3985.9 Da. Since each reduction of a disulfide bond increases the mass of a peptide by 2 Da, the peptide contains three disulfide linkages based upon the 6 Da mass shift.
  • Amino acid composition analyses were performed using an amino acid analyzer (Applied Biosystems 420H, Foster City, CA). Data normalization was done with respect to leucine. No discrepancies (excluding those residues which are either partially or totally destroyed during hydrolysis) in residue/mol values were recorded with respect to the Edman N-terminal sequencing analysis.
  • T ⁇ at position 31 of GsAF II is based upon amino acid compositional data and ES-MS analysis.
  • the unaccounted mass difference between the calculated mass value for the Edman deduced sequence and the mass spectral analysis for the native peptide is 186 Da assuming a free acid carboxyl terminus or 187 Da if the carboxyl terminus is amidated.
  • UV spectroscopy analyses of native GsAF II preparations indicate that the venom concentration of this peptide is approximately 3-5 mM.
  • This test measures the time interval required for a rat to withdraw its tail, via a spinally mediated reflex mechanism, from a high intensity light source (LITC Inc./ Life Sciences Instruments, Woodland Hills, CA 91367) focally applied to the dorsal surface of the appendage.
  • the intensity of the light beam has been experimentally defined such that naive animals will withdraw their tails within 2 to 4 seconds.
  • a maximum cut off time for the light source has been set at ten seconds to reduce the amount of secondary tissue damage. Data is expressed either as absolute time or a percentage of the maximal possible effect (MPE) which is described by the following equation where 10 seconds is the maximum: 21
  • % MPE (post-treatment latency - pretreatment latency )
  • peptide GsAF I was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA 01887). I.th. injections were made into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm using an extinction coefficient of 18710. Injection volume was 10 microhters. The injection vehicle was saline or 0.1% bovine serum albumin(BSA)/saline.
  • BSA bovine serum albumin
  • the rats were pretreated with GsAF I 30 minutes prior to exposure to the light source. Complete inhibition of the tail flick response (i.e., latency value greater than 10 seconds) was recorded in most rats following administration of 180 picomoles (666 nanograms) of GsAF I. A 95% MPE was attained for this dose and confounding side effects such as motor disturbances, limb impairment/paralysis, righting reflex, sedation, etc.) were either minimal or not present. Logarithmic decreases in the dose resulted in rapid loss of effect. 18 picomoles ( 66 nanograms) of GsAF I produced 29% MPE and 1.8 picomoles (6.6 nanograms) was inactive. Maximal activity was detected with a 30 minute pretreatment time. GsAF II Administration:
  • peptide GsAF II was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA)) into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm using the deduced molar extinction coefficient of 24310. Injection volume was 10 microhters. The injection vehicle was saline or 0.1% bovine serum albumin(BSA)/saline.
  • BSA bovine serum albumin
  • peptide GsAF I was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm using an extinction coefficient of 18710. Injection volume was 10 microhters. The injection vehicle was saline or 0.1 %bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF I 30 minutes prior to exposure to heat.
  • peptide GsAF II was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm using an extinction coefficient of 18710. Injection volume was 10 microhters. The injection vehicle was saline or 0.1% bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF LI 15 minutes prior to exposure to heat. A 100 % MPE was recorded for all animals receiving a 2.33 nanomole
  • Example 6 Analgesic Evaluation - Formalin Pain Test
  • the noxious stimulus for this test is the sub-cutaneous injection of a 5% solution of formalin into the dorsal surface of one of the hindlimbs of the animal.
  • Motor activity indices used in this test are 1) the total time spent licking that appendage and 2) the total number of flinching/shaking responses of the affected appendage.
  • Data collection is initiated immediately upon injection of the formalin solution into the limb.
  • the acute phase response is defined by the time interval of 0-5 minutes post formalin injection.
  • the tonic phase response is defined by the interval of 20-35 minutes post formalin injection
  • Data collection is done in a computerized format. Expression of the data is done using either absolute values or as percent control which is defined by the level of response following injection of saline vehicle.
  • peptide GsAF I was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm using an extinction coefficient of 18710. Injection volume was 10 microhters. The injection vehicle was saline or 0.1% bovine serum 25 albumin(BSA)/saline. The rats were pretreated with GsAF I 30 minutes prior to injection with the formalin solution..
  • peptide GsAF II was injected intrathecally (i.th.) into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) into the spinal subarachnoid space between lumbar spinous processes L4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels were based upon concentrations deduced from ultraviolet absorbance values at 280 nm as stated previously. Based upon a mass of 3980 Da, 583 pmoles corresponds to 2.3 micrograms of GsAF II, and 2.33 nmoles corresponds to 9.3 micrograms of GsAF II. Injection volume was 10 microhters. The injection vehicle was saline or 0.1% bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF II 15 minutes prior to injection with formalin.
  • BSA bovine serum albumin
  • GsAF I was retained. Tonic flinch response was inhibited 86% (i.e. 14% of control) and tonic lick duration was reduced 91% (i.e. 9% of control). This property has only been reported for strong analgesic compounds that interact with ⁇ -opioid receptors. It also demonstrates that the analgesic activity of GsAF I is not dependent upon the interruption of the initial rapid firing of sensory fibers (primarily c-fibers) or occlusion of wind-up within dorsal horn neurons.
  • Tissue injury results in inflammation and hyperalgesia (i.e. increased magnitude or duration of pain response to supra threshold noxious stimuli) at both the site of injury and at adjacent tissue sites.
  • hyperalgesia i.e. increased magnitude or duration of pain response to supra threshold noxious stimuli
  • paw withdrawal latencies were determined in adult 350-400 gram male Sprague Dawley rats following the unilateral injection of carrageenan, a seaweed extract, into the hindpaw in accordance with the method of Hargreaves et al, Pain, 32:77-88, 1988. Briefly, withdrawal latencies are measured by placing the rat on a glass plate and focusing radiant heat from the underside of the plate toward the hindpaw surface.
  • Latencies values are recorded in seconds to withdrawal of the hindpaw from the surface of the plate. Basal measurements are made prior to injection of the carrageenan (4 mg/hindpaw), followed by a measurement at 150 min post carrageenan injection to obtain the level of hyperalgesic response. Subsequent to the second measurement, GsAF II or vehicle (i.e.0.1% BSA saline) is administered through an indwelling intrathecal cannula positioned within the lumbar enlargement of the spinal cord. Anti-hyperalgesic activity is determined by measuring paw withdrawal latencies at various time intervals following compound administration. Concurrent with the paw withdrawal latencies, physical measurements of paw volume and paw temperature are recorded to detect anti- inflammatory and anti-pyretic activities.
  • GsAF II or vehicle i.e.0.1% BSA saline
  • GsAF II is an effective analgesic/anti- hyperalgesic compound for acute peripheral inflammatory pain. GsAF II does not appear to possess anti-inflammatory properties since the induction of analgesia was not associated with an acute reduction of edema or of body temperature.

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Abstract

L'invention porte sur de nouvelles méthodes de traitement de la douleur consistant à administrer à un mammifère le nécessitant une dose à effet analgésique d'un peptide présentant soit la séquence d'acides aminés: Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2 (SEQ ID NO:1), soit la séquence d'acides aminés: Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Leu-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Ile-Glu-Trp (SEQ ID NO:2. L'invention porte également sur un peptide purifié présentant la séquence d'acides aminés SEQ ID NO:1. Les peptides des séquences SEQ ID NO:1 et SEQ ID NO:2 peuvent de plus être utilisés dans des méthodes d'identification de composés analgésiogènes.
EP98905534A 1998-02-20 1998-02-20 PEPTIDES ANALGESIQUES EXTRAITS DU VENIN DE LA $i(GRAMMOSTOLA SPATULATA) ET LEUR UTILISATION Withdrawn EP1054896A1 (fr)

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FR2940973B1 (fr) * 2009-01-15 2013-05-24 Centre Nat Rech Scient Identification de nouvelles toxines antagonistes de canaux calcique type-t a visee analgesique
AU2012300197B2 (en) * 2011-08-24 2018-01-18 The University Of Queensland Pest-controlling agents isolated from spider venom and uses thereof
WO2023215528A1 (fr) * 2022-05-04 2023-11-09 University Of Rochester Utilisation de gsmtx4 et de conjugués de gsmtx4 pour améliorer la guérison après une blessure articulaire

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US5756663A (en) * 1996-01-03 1998-05-26 Zeneca Limited Antiarrhythmic peptide from venom of spider Grammostola spatulata

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US6071970A (en) * 1993-02-08 2000-06-06 Nps Pharmaceuticals, Inc. Compounds active at a novel site on receptor-operated calcium channels useful for treatment of neurological disorders and diseases
EP1336409B1 (fr) * 1995-06-27 2007-04-11 Eisai R&D Management Co., Ltd. Compositions et formulations permettant de produire une analgésie et d'inhiber la progression de troubles liés à des douleurs neuropathiques

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US5756663A (en) * 1996-01-03 1998-05-26 Zeneca Limited Antiarrhythmic peptide from venom of spider Grammostola spatulata

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