[go: up one dir, main page]

WO1989002920A2 - Article pour rendre inactifs des materiaux toxiques - Google Patents

Article pour rendre inactifs des materiaux toxiques

Info

Publication number
WO1989002920A2
WO1989002920A2 PCT/US1988/003422 US8803422W WO8902920A2 WO 1989002920 A2 WO1989002920 A2 WO 1989002920A2 US 8803422 W US8803422 W US 8803422W WO 8902920 A2 WO8902920 A2 WO 8902920A2
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
article
carrier
enzyme
ser
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.)
Ceased
Application number
PCT/US1988/003422
Other languages
English (en)
Other versions
WO1989002920A1 (fr
Inventor
Richard F. Taylor
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.)
Arthur D Little Inc
Original Assignee
Arthur D Little Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Arthur D Little Inc filed Critical Arthur D Little Inc
Publication of WO1989002920A2 publication Critical patent/WO1989002920A2/fr
Publication of WO1989002920A1 publication Critical patent/WO1989002920A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Definitions

  • compositions in which one or more chemicals, including enzymes, are bound to support materials for use in the controlled release of the chemicals have described compositions in which one or more chemicals, including enzymes, are bound to support materials for use in the controlled release of the chemicals.
  • one or more chemicals including enzymes
  • Hartman describes an enzyme-impregnated carrier
  • U.S. Patent No. 3,551,556 Kliment teaches controlled release of antibiotics and other chemotherapeutics from a polymeric carrier.
  • the present invention relates to an article for the inactivation or detoxification of a target ligand, which is a toxic material or disease-causing agent, as well as to methods of making and using the article.
  • the article comprises at least two receptors, both of which bind the target ligand, and a carrier, which is a solid material.
  • the first receptor (referred to as the binding receptor), binds the target ligand and the second receptor (referred to as the degradative receptor), in addition to binding the target ligand, acts upon the target ligand in such a manner as to cause its inactivation, generally by degrading it.
  • a target ligand may become attached to both a binding receptor and a degradative receptor.
  • both types of receptors may be in sufficiently close proximity to one another that a single target ligand interacts with both a binding receptor and a degradative receptor.
  • the two receptors act independently so that each will inactivate a separate target ligand.
  • the present invention also relates to the composition of novel peptides, useful in the article, that will serve as receptors. These novel peptides may be produced synthetically or by recombinant DNA procedures.
  • the article of the present invention comprises a first receptor, which is a protein that binds the target ligand, a second receptor that is an enzyme which specifically acts upon the target ligand, resulting in its degradation, and a carrier to which both receptors are affixed.
  • the article is comprised of a solid supporting material, to which is affixed at least two proteinaceous receptors: a first receptor, which specifically binds a toxic material to be inactivated, and a second receptor, which is an enzyme that hydrolyzes the toxic ligand, rendering it inactive.
  • a target ligand which is generally a toxic material
  • both receptors are affixed to a solid carrier, by being immobilized on its surface(s), incorporated into it as it is produced, or otherwise attached.
  • a proteinaceous membrane film such as bovine serum albumin co-polymerized with glutaraldehyde
  • at least one binding receptor such as acetylcholinesterase (AchE)
  • at least one degradative receptor such as an organophosphorusdegrading enzyme, which degrades the target ligand.
  • the carrier containing the receptors is then further processed into materials, such as bandages, sheets, beads, powders or gels.
  • Figure 1 is a schematic representation of one embodiment of the invention illustrating the use of AchE as the binding receptor and a hydrolytic enzyme as the degradative receptor.
  • Figure 2 is a graphic representation of the rate of hydrolysis of an organophosphorus toxin by free and immobilized rat liver enzyme.
  • Figure 3 represents the temporal changes in AchE protecting activity of BSA-membrane as a function of storage temperature.
  • Figure 4 represents the changes in AchE-protecting activity of BSA and PVC-silica membranes as a function of pH.
  • Figure 5 represents the changes in AchE-protecting activity of BSA and PVC-silica membranes.
  • Figure 6 represents the changes in AchE-protecting activity of BSA and PVC-silica membranes immersed in blood solutions of various concentrations and suspended in soil slurries of various concentrations.
  • the present invention relates to an article for use in inactivating a target material, which is generally a toxic material or an invasive microbe, as well as to novel peptides useful in the article, a method of making the article and a method of using the article.
  • a target material which is generally a toxic material or an invasive microbe
  • novel peptides useful in the article a method of making the article and a method of using the article.
  • the term "inactivate” as used in this regard describes any means by which a target ligand is bound, degraded, decomposed or otherwise rendered nontoxic by the receptor ligands.
  • the article and method of using it are particularly valuable in inactivating or detoxifying toxic materials encountered in environmental, chemical, military and industrial settings.
  • the article of the present invention comprises at least two receptors and a carrier to which the receptors are affixed.
  • One receptor (referred to as a binding receptor) binds a target material, which is referred to herein as a target ligand, which is to be inactivated, and the second receptor (referred to as a degrading receptor) is a substance, such as an enzyme, which degrades the target ligand.
  • Receptors which bind the target ligand to be inactivated contain at least one binding site that forms a bond with at least one binding site of the target material. Contact between the binding receptor and the target ligand results in binding of the target ligand by the receptor. Binding between ligand and receptor may be accomplished, for example, by covalent, ionic or hydrogen bonding or by Van der Waals forces. The receptor binds the target ligand and eliminates it from further contact with the surface to be protected.
  • Binding receptors suitable for use in the article of the present invention include proteins (such as enzymes), glycoproteins, biological receptors (such as neural receptors), lectins, antibodies and peptides.
  • at least one esterase protein such as acetylcholinesterase, aldolase, trypsin, alpha-chymotryps ⁇ n, butyrylcholinesterase, alliesterase, alkaline phosphatase, kallikrein or subtilisin, is used.
  • Plant sulfhydryl esterases such as papain, chymopapain, ficin, and bromelain are also useful.
  • peptides modelled after the active sites of naturallyoccurring enzymes such as AchE and papain can be used as binding receptors.
  • Such peptides modelled after active sites and receptors such as those described above can be synthesized mechanically or produced using recombinant DNA methodology that is known in the art. Examples of methods useful in producing such receptors are described in Carter, P. and J. Wells, Engineered Enzyme Specificity by Substrate-Assisted Catalysis, Science, 237:394-399 (1987); Carter, P., Biochemical Journal, 237: 1 (1986); Stewart, J. M. and J.D. Young, Solid Phase Peptide Synthesis, (2nd ed.), Pierce Chemical Co., Rockford, Illinois and Maniatis, T. et al., Molecu lar Cloning, Cold Spring Harbor, 1982, the teachings of which are incorporated herein by reference.
  • the degrading receptor will generally be an enzyme and can act on the target ligand in one or more ways. For example, it may bind the target ligand in such a way that the susceptible bond is brought into close proximity to the catalytic group on the enzyme's active site.
  • the enzyme may combine with the target to form an unstable, and more readily degraded covalent intermediate.
  • the enzyme may bring about acid or base catalysis by donating or accepting protons.
  • the enzyme may also induce conformational changes in the structure of the target material, making the chemical bonds within the target easier to break.
  • useful degrading receptors include enzymes, such as oxidoreductases and hydrolases.
  • enzymes such as carboxylic ester hydrolases, thiolester hydrolases, phosphate monoester hydrolases, amino acid decarboxylases, L-amino acid oxidases and D-amino acid oxidases can be used.
  • Degrading ligands useful in the article of the present invention can be naturally-occurring (e.g., enzymes isolated from their natural source). They can also be produced using genetic engineering methods or can be synthesized mechanically. Examples of methods useful in producing such ligands are described by P. Carter and J. Wells in Engineered Enzyme Specificity by Substrate ⁇
  • any solid material capable of supporting the binding receptor and the degrading receptor, and to which the receptors can be affixed is suitable for use in the article of the present invention.
  • the solid support can be, for example, in the form of woven fabric (e.g., sheets, bandages, etc.), porous foam pads, .absorbent membranes, solvent-based porous elastomers, or beads.
  • the binding receptor and the degrading receptor can be affixed onto beads or solid particles, which are and then incorporated into a foam, gel or an aerosol.
  • Supports useful in the article can be obtained commercially in the form of silica-impregnated polyvinylchloride supports (product of Amerace
  • vinyl polymer beads polymeric beads comprised of agarose, dextran, cellulose, vinyl polymers, acrylates, acrylamides, polystyrene-divinylbenzene or polyethers (product of Pierce Chemical Co., EM Science, Rohm & Haas Co., Dextran Products Ltd., Toyo Soda Manufacturing Co., and others), and alumina or silica (glass) beads (product of Aluminum Company of America, Universal Adsorbants, ICN Adsorbants, W.R. Grace & Co., and others).
  • These materials to which the receptors have been affixed may be further processed into bandages, cloths, sheets or powders using techniques described below.
  • both the binding and the degrading receptors are affixed to the carrier.
  • "affixed” means fixed or fastened in any manner.
  • the binding receptor and the degrading receptor can be affixed to the surface of the carrier or they can be incorporated into the carrier (e.g., by being impregnated within the interstices of the carrier).
  • the manner in which the two receptors are affixed to the carrier will vary with the type of carrier used.
  • the article of this invention is designed in such a way that the carrier to which the receptors are affixed comes into contact with the target ligand.
  • contact means the touching, meeting, or juxtaposition of the receptor-bearing carrier and the target material in such a manner that the target material is bound to either one or both of the receptors. That is, the binding receptor and the degrading receptor are affixed to the carrier such that target ligand comes in contact with either the binding receptor, which acts to prevent further entry of the target material into the protected surface, the degrading receptor, which acts to degrade the target material, or both receptors.
  • the carrier containing the binding receptors and the degradative receptors is draped, wrapped, or otherwise placed over the area (e.g., skin surface, exterior surfaces of boxes containing food or beverage, etc.) to be protected and decontaminated.
  • the manner in which the article is applied will depend upon the carrier and may involve the use of a sprayer or atomizer to disperse the receptor-impregnated particles.
  • the article of this invention provides a means of inactivating toxic material, such as those encountered in an environmental, military, medical, chemical, or industrial context.
  • Toxic materials/ligands which can be inactivated' include, but are not limited to, materials acting as systemic poisons such as pesticides, nerve agents, bacteria, or viruses.
  • the toxin can be in solid or particulate form, as in the case of a bacterium or a particleadsorbed chemical.
  • the toxin can be in a liquid or gaseous phase, such as the organophosphorus compounds DFP (phosphorofluoridic acid bis(1-methylethyl) ester) and GB (methylphosphonofluoridic acid 1-methylethyl ester), or the carbamate compound Carbaryl (1-napthalenol methylcarbamate).
  • a solid carrier is made by forming a membrane comprised of bovine serum albumin co-polymerized with glutaraldehyde.
  • macroporous supports of silica-impregnated polyvinylchloride are used (e.g., Macroporous Plastic Sheets, Amerace Corp.).
  • solid beads or irregular particles comprised of inorganic material (e.g., alumina or silica) or polymers (e.g., agarose, cellulose, dextran, acrylate, styrene, and others) are used.
  • acetylcholinesterase can be immobilized onto the carrier to serve as the binding receptor.
  • Preparations of rat liver organophosphorous-degrading enzyme can be immobilized onto the same carrier to serve as the enzymatic receptor.
  • Example II Preparation of receptor-containing membranes is described in detail in Example I. Briefly, this is carried out as follows: A mixture of binding receptors, degradative receptors and serum albumin is prepared; the mixture is allowed to polymerize with buffered glutaraldehyde. Preparation of silica-impregnated PVC supports, microporous plastic sheets (MPS), activated by treatment with polyethyleneimine (Amerace Corp.) is described in Example II. The sheets are coated with glutaraldehyde in solution containing receptors .
  • MPS microporous plastic sheets
  • a receptor substance-containing bead support is described in Example 3. As described, a predetermined amount of receptor substance is reacted with a preactivated bead in an appropriate buffer for 4 to 24 hours at room temperature.
  • the described membranes and MPS preparations can be used directly to bind and destroy toxic target materials, or may be attached to an adhesive backing or similar material to form a bandage.
  • the receptor substance-containing beads can be used directly for similar applications or formulated as a salve, gel, cream or lotion.
  • a two-compartment bag or soft metal tube is used. One compartment contains dry, receptor substance-containing beads and the second compartment contains a gel or foam under pressure. For use, a separator between the two compartments is removed or broken. The contents of the two compartments are mixed and the mixture is expelled from the bag or tube through a nozzle onto the surface to be protected.
  • Membranes This example illustrates the preparation of membranes made from receptor-impregnated glutaraldehyde.
  • a mixture is prepared containing from 0.1 to 1.0g of bovine or human serum albumin and a predetermined amount of receptor substance in 5 to 10 ml of an appropriate buffer at pH 6.5 to 7.5 (e.g., 0.01 to 0.1M KH 2 PO 4 -K 2 HPO 4 or 0.005 to 0.1m Tris-HCL containing 0.4M KCl and 0.05M NaCl. From 0.1 to 5% (by volume) gluraraldehyde is added to the mixture, which is mixed gently at room temperature for 5 to 30 minutes.
  • the mixture is spread evenly onto glass plates and allowed to polymerize and age for 10 to 16 hours.
  • the resulting membrane is floated off the glass plate using water immersion, and dried. It is stored dry until used.
  • the amounts of albumin, glutaraldehyde and buffer are varied to alter polymerization time and density of the membrane.
  • the amount of receptor substance to be used depends on its purity and binding activity. For example, from 0.01 to 200mg of enzymes such as acetylcholinesterase, butylrylcholinesterase, trypsin, alpha-chymotrypsin, subtilisin, papain, and toxic agent hydrolysis enzymes are used.
  • Example II Preparation of Receptor-Containing Microporous Polymer Sheets
  • Squares (3 ⁇ 3 cm) of the MPS are wetted in 0.05M NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 7) for 1 to 5 minutes, and then immersed into a solution of 5% glutaraldehyde (by volume) in the same buffer for 20 to 60 minutes at room temperature.
  • the glutaraldehyde-covered MPS squares are placed into 5 to 10 ml of buffer containing a predetermined amount of receptor substance for 12 to 20 hours at 4°C. After this time, the MPS support (now containing immobilized receptor substance) is washed, dried and stored dry until used.
  • Example III Preparation of Receptor-Containing Beads
  • a predetermined amount of receptor substance is reacted with a bead at room temperature, under the reaction conditions which depend on the support used.
  • 1 to 50 mg of receptor substances such as acetylcholinesterase and the organophosphorus-degrading enzymes from rat liver, squid hepatopancreas, Escherichia coli or Tetrahymena thermophilia were each reacted with 20 ml (approx.
  • Reacti-Gel HW-65F a vinylpolymer based, 30 to 60 micron bead activated with carbonyldiimidazole (Pierce Chemical Co.) in 1M NaHCO 3 -Na 2 CO 3 buffer, pH 10 for 12 to 18 hours at room temperature.
  • the resulting beads were washed, dried and stored, either wet or dry, until used.
  • activated supports can also be used, such as carbodiimidazoleactivated agarose, dextran and glass beads (Pierce Chemical Co.); epoxy-, N-hydroxysuccinimide- or cyanogen bromide activated agaroses (Pharmacia Fine Chemicals and BioRad Laboratories); or epoxyactivated acrylates and hydroxymetacrylates (Rohm Pharma and Dextran Products).
  • Example IV Ability of Binding Receptor to Protect Against Toxic Materials This example illustrates the ability of a known amount of binding receptor in the form of a protein or peptide to protect AchE against inhibition by a target ligand.
  • Binding receptors are available commercially (Sigma Chemical Co., St. Louis) with purities of 90% or better. No further purification is needed.
  • Acetylcholinesterase can also be prepared from electric eel (Electrophorus sp.) electroplax by homogenizing 50g of minced tissue in 1 mM EDTA. The resulting filtrate is centrifuged at 20,000 ⁇ g at 4oC and the crude membrane preparation homogenized again for 30 seconds in 100 ml of 2 mM
  • Example V Use of Synthetic Peptides as Binding Receptors
  • Synthetic peptides able to bind to a wide range of alkylating agents can be made by procedures well known in the art.
  • Synthetic peptides modelled after the active sites of the binding receptors of Example I can also be used as binding receptors.
  • a commercially-available solid-state peptide synthesis procedure is employed. In this procedure, a 4-methylbenzhydrylamine resin is reacted with amino acids using carbodiimide coupling. The resin (2g per run) is reacted with 6g. each of amino acid and carbodiimide per step.
  • the peptide is deblocked and removed from the support by hydrofluoric acid (HF) treatment and purified/characterized by high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • a reversephase 10 micron Vydac TP218 preparative HPLC column (22.5 ⁇ 250 mm) is used with 10 minute isocratic elution (0.1% aqueous trifluoroacetic acid) followed by a 20 min. linear gradient of 0 to 75% acetonitrile in trifluoroacetic acid.
  • Purified peptides are collected as they are eluted from the column and lyophilized. The process generates a series of peptides with primarily hydrophobic, acidic amino acids in a sequence similar to the papain active site sequence of
  • peptides P13-P18 histidine (His) is added and phenylalanine (Phe) substituted for Tryptophan (Trp) to vary the hydrophobic character of the peptides.
  • His histidine
  • Phe phenylalanine substituted for Tryptophan
  • Trp Tryptophan
  • Example VI Preparation of Degradative Receptors for the Hydrolysis of Organophosphorous Compounds
  • the following example describes preparation and degradative capacity of various receptors that can be used in the article of the present invention for the hydrolysis of organophosphorus compounds.
  • Hydrolytic enzyme is available from several sources. Batch cultures of E. coli (ATCC #25922) are maintained on Tryptic Soy Broth at 37°C and the cells in log phase collected by centrifugation, washed with sterile saline, and stored at -20°C. The protozoan Tetrahymena thermophilia (a gift from Dr. Wayne Landis, U.S. Army, CRDEC, Aberdeen) is maintained on broth containing 10g proteose peptone and 3g yeast extract per liter. These cells are harvested by centrifugation at 1000 ⁇ g to prevent cell rupture and washed with 0-9% saline. Squid (Loligo sp.) hepatopancreas and rat liver are obtained fresh.
  • Extractions are carried out in 2-5 volumes of 5mM Tris-HCl (pH 7.2) containing 400 mM KCl and 50 mM Nacl (hereinafter Buffer 1). Extracts of rat liver and squid hepatopancreas are briefly homogenized and then centrifuged at 20,000 ⁇ g. T. thermophila will lyse significantly even during harvesting. E. coli must be pretreated with lysozyme (20-50 ug/ml for 15 minutes) so that sonication will result in quantitative cell destruction and enzyme extraction.
  • Extracts are first purified on a Sephadex G-25 column and eluted with 5mM Tris-HCl containing 400 mM KCl and 50 mM NaCl.
  • the enzyme-containing fraction is then concentrated in volume using an ultrafilter and applied to a DEAE ion exchange column packed with 5 mMTris-HCl, pH8.
  • Enzyme is eluted from the ion exchange column with a 0-0.3 M NaCl gradient in the packing buffer. Amounts of protein are assayed at each step using the Lowry procedure. O.H. Lowry, N.J. Rosenbrough, A.L. Farr and R.J. Randall, Journal of Biological Chemistry, 193 :265 (1951).
  • Enzyme activity can be determined in a variety of ways, depending on the reaction products to be expected.
  • fluoride ion liberation from substrates such as DFP (Phosphorofluoridic acid bis (1-Methylethyl) ester
  • DFP Phosphorofluoridic acid bis (1-Methylethyl) ester
  • Oxygen F Electrode Model 96-09 selective fluoride ion electrode
  • Hydrolysis of DFP, GD, and GB occurred with all the above-mentioned enzyme preparations (Table 4).
  • the procedures of Example I were used to test the ability of these hydrolytic ligands to protect AchE from attack by the target ligand.
  • Microgram amounts of the hydrolysis enzymes can protect AchE from being inactivated by a variety of organophosphorus compounds (Table 5).
  • AchE is also protected against the phosphonothionate VX (Methylphosphonothioic acid S-[2-[bis(1-methylethyl)amino]ethyl]o-ethyl ester). Since these particular hydrolytic receptors should not degrade the phosphothionate VX, they are most likely merely binding to VX. The net effect on protecting AchE from the target ligand is, however, similar. This exemplifies the ability of this invention to inactivate toxic ligands by means other than physical degradation or decomposition of the target ligand.
  • VX Metal nothionate VX
  • Agent concentration was 10 -3 M.
  • the data are averages of 4 to 6 determinations with each agent and enzyme for each of 3 to 4 separate extractions.
  • thermophila T. thermophila
  • Example V See Example V for enzyme preparation.
  • Example VII Use of Solid Carriers to Protect Against Target Ligands This example illustrates the ability of several solid matrices to immobilize the binding and degradative receptors and to protect AchE from attack by target substances.
  • Membrane films, MPS sheets and vinylpolymer beads were prepared according to the methods described previously. Approximately 10 mg of protein-bound bovine serum albumin membrane, 1 cm 2 of Amerace or 2ml of vinylpolymer beads were soaked in 0.5 ml of neutral buffer and assayed for the ability to protect AchE from a target chemical (see Example I for details). Bovine serum albumin membranes, MPS sheets and polymer beads, when impregnated with various binding and degradative receptors, protect AchE from inhibition by toxic organophosphorus compounds (Tables 6 and 7).
  • thermonhila Enzyme Crude 810 - 15 43 46 G-25 196 - 0 (7) 20
  • thermonhila T. thermonhila
  • thermophilia (0.20) 0 (-100) 0 (-100)
  • Example VIII Ability of Immobilized Receptors to Degrade Target Materials This example illustrates that, although the immobilized enzymes have a decreased absolute specific activity as compared to the free enzyme, their altered reaction kinetics result in more total ligand hydrolysed than the free enzyme preparation.
  • Rat liver enzyme was purified and assayed as described previously.
  • the agent (GD) (10 -3M) was reacted in a 5 ml volume of buffer 1 with 13 mg of free enzyme, and an amount of receptor equivalent to 4, 2, or 0.3 mg enzyme immobilized on serum albumin membranes, Silica-PVC, or Reacti-Gel, respectively.
  • Figure 1 shows that hydrolysis of GD approached steady-state after 30 minutes in the free enzyme preparation, but degradation of GD continued to increase beyond this time in the immobilized preparations. Thus, more ligand is inactivated in the immobilized preparations than would be the case if the receptors were not immobilized.
  • Example X Non-toxicity of Solid Carriers and Immobilized Receptor Preparations This example illustrates that membrane-based and MPS based dressings containing immobilized acetylcholinesterase (AChE) and rat liver hydrolysis enzyme are non-toxic to laboratory animals.
  • AhE immobilized acetylcholinesterase
  • rat liver hydrolysis enzyme are non-toxic to laboratory animals.
  • 8 mm (Miltex 33-28 biopsy punch) wounds were made in the shaved, upper right flanks of ten Hartley guinea pigs (250-350g) anesthetized with sodium nebutal, i.p., at 30 mg/kg.
  • the bandage preparations 1 cm 2 backed onto Johnson & Johnson
  • Dermicel first aid tape were securely placed over the wound and allowed to remain on the wound for 30 minutes. After this time, the bandage was removed and the wound covered with a sterile band-aid. This procedure was repeated for 5 weeks, wounding in a new area each week. The animals were kept under routine observation and the wounds were examined daily. Blood samples of 1.5 ml from the ocular vein were collected from each animal at weeks 4 and 5 for immunoanalysis of plasma to detect any circulating antibody to the bandage protein components. During the course of our 5 week study, no obvious morphological changes occurred in the animals. The wounds healed normally and the animals gained weight at a normal rate. Aside from any anesthetic problems, there were no signs of distress or shock in the animals.
  • the controls showed distinct, clear precipitin lines while no lines were seen before or after staining between any of the plasma samples and any bandage component. Repeated application of the bandages does not lead to an immune response and the immobilized BSA membrane and Amerace MPS dressings present no toxic or allergic hazard as determined in test animals.
  • This example illustrates the removal of DFP, malathion and carbaryl (Sevin) from wounds and skin under real time conditions in laboratory animals.
  • Guinea pigs (approximately 700 to 800 g) were anesthetized with nebutal (30 mg/Kg), shaved and wounded with an 8 mm skin biopsy punch (see Example X for details).
  • the wound was filled with either 50 ul of neat DFP, 50 ul of neat malathion, or a 1 to 4 dilution of carbaryl in ethanol (approximately 6.3 mg).
  • agent was immediately washed out of the wound (pH 4 water, approximately 10 ml/wash) to act as a 0 time control.
  • Example XII Stability of the Solid Carriers and Immobilized-Enzyme Preparations This example demonstrates that BSA and MPS-based membrane preparations are stable for long periods under a variety of environmental conditions. During a two-week period, only high temperature (50°C) caused any decrease in AchE protecting activity in BSA membranes ( Figure 3).

Abstract

Un article utile pour rendre inactifs des matériaux toxiques comprend un support solide auquel son liés au moins deux types de récepteurs. Ces récepteurs agissent sur le matériau visé en s'y liant et en le dégradant, le rendant inoffensif par rapport à la surface que l'on veut protéger. Le support solide est particulièrement utile sous forme de pansement ou d'un drap avec lequel on peut recouvrir des équipements, des denrées alimentaires, ou des personnes. De nouveaux peptides utilisés dans cet article peuvent servir de récepteurs des toxines visées.
PCT/US1988/003422 1987-10-05 1988-10-04 Article pour rendre inactifs des materiaux toxiques Ceased WO1989002920A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10531287A 1987-10-05 1987-10-05
US105,312 1993-08-10

Publications (2)

Publication Number Publication Date
WO1989002920A2 true WO1989002920A2 (fr) 1989-04-06
WO1989002920A1 WO1989002920A1 (fr) 1989-04-06

Family

ID=22305122

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1988/003422 Ceased WO1989002920A1 (fr) 1987-10-05 1988-10-04 Article pour rendre inactifs des materiaux toxiques

Country Status (2)

Country Link
EP (1) EP0381701A1 (fr)
WO (1) WO1989002920A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPM334794A0 (en) * 1994-01-13 1994-02-03 Commonwealth Scientific And Industrial Research Organisation Enzyme based bioremediation
US9538390B2 (en) 2015-01-23 2017-01-03 Microsoft Technology Licensing, Llc Wireless connectivity using white spaces

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3809617A (en) * 1972-11-15 1974-05-07 American Cyanamid Co Device for detecting anticholinesterase materials
FR2558171B1 (fr) * 1984-01-12 1986-08-01 Oreal Materiau polymerique a action enzymatique, son procede de preparation et son utilisation en tant que medicament
US4576817A (en) * 1984-06-07 1986-03-18 Laclede Professional Products, Inc. Enzymatic bandages and pads
DE3606265A1 (de) * 1986-02-27 1987-09-03 Roehm Pharma Gmbh Wundauflage auf polysaccharidbasis als traeger therapeutisch wirksamer, nicht-immobilisierter enzyme und mit hoher saugfaehigkeit

Similar Documents

Publication Publication Date Title
US4004979A (en) Preparation of active proteins cross-linked to inactive proteins
US4970156A (en) Immobilization of active protein by cross-linking to inactive protein
Nickerson et al. Keratinase: I. Properties of the enzyme conjugate elaborated by Streptomyces fradiae
Chubb et al. The enkephalins are amongst the peptides hydrolyzed by purified acetylcholinesterase
US4464468A (en) Immobilization of active protein by cross-linking to inactive protein
Abuchowski et al. Preparation and properties of polyethylene glycol-trypsin adducts
Hooper et al. Isolation of two differentially glycosylated forms of peptidyl-dipeptidase A (angiotensin converting enzyme) from pig brain: a re-evaluation of their role in neuropeptide metabolism
Beynon et al. Purification and characterization of a metallo-endoproteinase from mouse kidney
EP0313346B1 (fr) Cystéine protéases, production et utilisation
Gordon et al. Organophosphate skin decontamination using immobilized enzymes
Chestukhina et al. Crystal-forming proteins of Bacillus thuringiensis. The limited hydrolysis by endogeneous proteinases as a cause of their apparent multiplicity
Nielsen et al. . beta.-Lactamase III of Bacillus cereus 569: membrane lipoprotein and secreted protein
Rosenberg et al. Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups
EP1345634A2 (fr) Elimination de proteases ciblees a l'aide de pansements proteiques contenant des facteurs de croissance
Chao et al. Studies on rat renal cortical cell kallikrein. II. Identification of kallikrein as an ecto-enzyme
WO1989002920A2 (fr) Article pour rendre inactifs des materiaux toxiques
WO1989002920A1 (fr) Article pour rendre inactifs des materiaux toxiques
JP2832129B2 (ja) 酵素的分解によるタンパク質の切断方法およびその利用方法
DeFrank Organophosphorus cholinesterase inhibitors: detoxification by microbial enzymes
Leduc et al. Structure of the Cell Wall of Bacillus Species CIP 76‐111
Haskå Activity of bacteriolytic enzymes adsorbed to clays
Bendtzen Human leukocyte migration inhibitory factor (LIF) III. Further investigations on the serine protease nature of this lymphokine and its preference for arginine amides
Simmons et al. Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin
Evans Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)
Isobe et al. Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis