WO1987007531A1 - Elimination d'endotoxines - Google Patents

Elimination d'endotoxines Download PDF

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Publication number
WO1987007531A1
WO1987007531A1 PCT/GB1987/000406 GB8700406W WO8707531A1 WO 1987007531 A1 WO1987007531 A1 WO 1987007531A1 GB 8700406 W GB8700406 W GB 8700406W WO 8707531 A1 WO8707531 A1 WO 8707531A1
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endotoxin
sorbent
solid phase
anticoagulant
polymyxin
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Jonathan Cohen
Calvin John Ryan
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Royal Postgraduate Medical School
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Royal Postgraduate Medical School
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures

Definitions

  • the present invention relates to the removal of endotoxin from endotoxin-containing fluids.
  • Endotoxin which is part of the cell wall of gram negative bacteria, is implicated in gram-negative
  • Endotoxin is also implicated in various other serious conditions including chronic and acute liver disease, chronic and acute renal disease, radiation sickness and heat stroke.
  • Polymyxfn B is a cyclic polypeptide antibiotic which, in addition to its antimicrobial activity, has been known for many years to have potent anti-endotoxin properties.
  • Polymyxin B (PB) binds stoichiometrically to endotoxin, and will neutralize many endotoxin-
  • polymyxin B has been linked to Sepharose (Trade tMark) and also to polystyrene fibres, and such systems have been found to be capable i_ vitro of removing endotoxin from endotoxin-containing solutions.
  • the use of fibre- linked polymyxin B has also been described in hae operfusion of endotoxin-treated dogs, with improved survival in the treated group.
  • the present invention is based on the surprising observation that plasmapheresis of endotoxin-containing blood can be carried out successfully if a solid phase support carrying immobilised polymyxin B is treated before use with an anticoagulant.
  • This observation is surprising because we have attempted to use polymyxin B linked to a solid phase support to remove endotoxin in vivo using the technigue of plasmapheresis, but such attempts have failed because of fibrin deposition.
  • This deposition generally starts in the sorbent column within 5 to 10 minutes of starting plasmapheresis and within 15 to 20 minutes the plasmapheresis apparatus as a whole becomes clogged to such an extent that it can no longer function effectively.
  • the fibrin is deposited throughout the plasmapheresis system, not just in the sorption column itself, and the deposition occurs despite the presence of the heparin present conventionally in the circulating plasma.
  • the present invention provides an endotoxin sorbent comprising polymyxin B immobilised on a solid phase support, the solid phase-polymyxin B sorbent having been treated with an anticoagulant capable of preventing or limiting the deposition of fibrin to block free anticoagulant binding sites.
  • Polymyxin B may be used as such or in the form of a- salt for example, the sulphate. Also there may be used a mixture of any two or more components selected from polymyxin B and salts thereof. Unless otherwise specified, the term "polymyxin B" is used herein to encompass all such possibilities.
  • the solid phase support may comprise any substance capable of immobilizing polymyxin B. It is thought that in some cases at least polymyxin B can be bound to a support via an a ino group, and on this basis the support may be, for example, a solid material capable of forming a covalent bond with an amino group.
  • the polymyxin B may be immobilised on a solid support either directly or via a coupling group. Accordingly, other suitable solid phase support materials are those capable of accepting coupling groups suitable for the immobilisation of polymyxin B. Examples of solid support materials are agarose and agarose derivatives, other carbohydrates, polystyrene and regenerated cellulose.
  • the solid phase support may be used in any of a variety of physical forms, such as, for example, those proposed for affinity chromatography in general, for example, beads, fibres, webs, membranes and hollow fibres.
  • Beads for example, agarose beads, may be used in columns.
  • the size of beads preferred is generally in the range of from 45 to 165 ⁇ , but larger or smaller beads may be used, longer columns generally being required with larger diameter beads.
  • Such bead size/column length parameters will be well-known to those skilled in the art of affinity chromatography.
  • Other physical forms of solid support for example, fibres, for example, polystyrene fibres, webs, membranes and hollow fibres may be used analogously to their use in affinity chromatography generally.
  • the anticoagulant used to treat the polymyxin B sorbent must be capable of preventing or limiting the deposition of fibrin, either directly, that is to say, by influencing the conversion of' fibrinogen to fibrin, or indirectly, that is to say, by influencing any one or more of the other steps involved in the intrinsic and extrinsic pathways of blood coagulation.
  • Heparin is widely used and known to be effective in preventing fibrin deposition. It is readily available, relatively inexpensive, and substantially free from side effects. Accordingly, heparin is a preferred anticoagulant for the present invention, but any other anticoagulant having the proper-ty of interfering with fibrin deposition may be used.
  • heparin-type category are, for example, low molecular weight heparin derivatives and other heparin derivatives.
  • Such derivatives may have advantages over native heparin, for example, with regard to the specificity of their anti-fibrin- deposition activity.
  • lower molecular weight derivatives may affect platelets less than native heparin does (see, for example, Salz an E. ., Rosenberg R.D., Smith .H., Linden J.N., and Savreau . f J. Clinical Investigation (1981) J55_ 64-67).
  • Other strongly acidic substances having a heparin-like anticoagulant effect may be used, for example, dextran sulphate. Hirudin may also be used.
  • Antithrombotic agents directed towards different aspects of the haemostatic process may be used as the anticoagulant, for example, inhibitors of intrinsic coagulation; and also platelet aggregation inhibitors (antiplatelet agents), for example, adenyl compounds, for example, AMP adenosine and 2-chloroadenosine; dipyridamole and lidoflazine; prostacyclin and prostaglandin E; anti-inflammatory drugs, for example, aspirin, phenylbutazone, indomethacin, meclofenamate and hydroxychloro ⁇ uine; membrane stabilizers, for example, local anaesthetics and many antihistamines; serotonin antagonists; sulphydryl inhibitors, for example, N-ethylmaleimide and p-hydroxymercuribenzoate; arginine esters and other guanidino compounds; various miscellaneous drugs which inhibit platelet aggregation, for example, clofibrate,
  • the present invention further provides a process for producing anticoagulant-treated, solid phase- immobilised polymyxin B, which comprises immobilising polymyxin B on a solid phase support and subsequently treating the solid phase support carrying polymyxin B with an anticoagulant capable of preventing or limiting fibrin deposition under conditions such that free anticoagulant binding sites are blocked.
  • the anticoagulant treatment of the solid component may be carried out by incubating a sample of the polymyxin B-bearing solid phase support with a pre ⁇ determined volume of a solution comprising the anticoagulant, or an anticoagulant-containing solution may be passed over and/or through a sample of the polymyxin B-bearing solid phase component.
  • polymyxin B may be immobilised on the support in a conventional manner.
  • agarose available commercially in bead form as "Sepharose”
  • the agarose is activated. for example, using cyanogen bromide, and then reacted with polymyxin B, for example, according to the method of Issekutz (J. Immunological Methods 6_1_ (1983) 275- 281).
  • the buffers and reagents should be prepared in a sterile fashion using pyrogen-free water, glass-ware should be rendered as pyrogen-free as possible, and sterile plasticware should be used whenever practicable.
  • Materials of the "Sepharose” type can be activated with cyanogen bromide in a conventional manner, or Sepharose itself can be obtained in activated form from the manufacturers (Pharmacia, Uppsala, Sweden) .
  • the activated material is generally swollen, washed, for example, in 0.1 M HC1, and suspended in a suitable buffer, for example, a sodium bicarbonate buffer, for- example, 0.1 M NaHCO- j containing 0.5 M NaCl, final pH 8.3.
  • a suitable buffer for example, a sodium bicarbonate buffer, for- example, 0.1 M NaHCO- j containing 0.5 M NaCl, final pH 8.3.
  • the resulting gel is preferably allowed to settle, excess buffer removed, and then polymyxin B, generally in the form of polymyxin B sulphate, is added to the gel, generally in the form of a solution in a buffer, for example, as described above.
  • the mixture of polymyxin B and the agarose material for example, Sepharose
  • a suitable time for example, overnight
  • a temperature generally between 0°C and room temperature, for example, in the range of from 2°C to 18°C, for example, 4°C.
  • Excess buffer is then generally discarded.
  • an agent capable of blocking remaining reactive groups for example, ethanolamine.
  • the polymyxin B may be immobilised in an analogous or conventional manner.
  • the immobilisation of polymyxin B on polystyrene fibres has been described by Endo .et_ a_l. (Abstracts of the 14th International Congress of Chemotherapy 1985, Kyoto, Japan, Abstract P-40-9, International Society of Chemotherapy, active halogen method) , and also by Hanasawa e_ s_ (Therapeutic Apheresis: A critical look. Nose Y,
  • treatment of the solid phase support on which polymyxin B has been immobilised with the anticoagulant may be carried out by incubating the solid component with a solution comprising the anti ⁇ coagulant.
  • a range of temperatures may be used, for example, from 2°C to 25°C, but room temperature is generally suitable.
  • the amount of anticoagulant used and the* time of incubation are such that free anticoagulant binding sites on the solid component are blocked.
  • the number of free anticoagulant binding sites will vary depending on the chemical constitution and physical form of the solid, and on the loading with polymyxin B. Having appreciated that the sites, and preferably as many as possible, should be blocked, it is a matter of simple trial and error to determine appropriate amounts of anticoagulant and incubation times.
  • this priming amount is generally within the range of from 5 to 20 units of heparin per ml of saline, for example, 10 units of heparin per ml.
  • Amounts of heparin as low as 100 units per ml of Sepharose gel will give satisfactory results for a short time but after 30 to 40 minutes, fibrin deposition in the extracorporeal circulation is observed. Accordingly, amounts of heparin in excess of 100 units per ml Sepharose gel are recommended, preferably 500 units or more, and especially 1,000 units or more, for example, 5,000 units or more, for example, 10,000 units.
  • heparin re ⁇ uired for other systems may be determined readily by simple trial and error but, as indicated above, it is generally convenient to • use an excess, and preferably a large excess, and these considerations apply, mutatis mutandis , to other chosen anticoagulants.
  • the solution may be passed over and/or through the batch of solid component.
  • the amount of anti ⁇ coagulant to be used may be determined by simple trial and error and the considerations given above with regard to the amount of anticoagulant to be used also apply to the present embodiment of the invention.
  • an excess, especially a large excess, of anticoagulants especially heparin compared with the amount generally incorporated in saline for the purpose of priming plasmapheresis apparatus before use.
  • the guide values given above for heparin may be followed.
  • the perfusion of the polymyxin B-bearing solid phase support with anticoagulant-containing solution may be carried out as a separate step before incorporation of the treated material in plasmapheresis apparatus, or the perfusion may be carried out _in_ situ in plasmapheresis apparatus before use, that is to say, before connection to the subject to be treated.
  • the resulting anticoagulant-treated endotoxin sorbent may be prepared in advance and stored before use, for example, in pyrogen-free water containing a suitable preservative, for example, 25% (v/v) ethanol, or 0.2% (w/v) sodium azide, or it may be prepared as required, for example, _in_ situ as described above.
  • a suitable preservative for example, 25% (v/v) ethanol, or 0.2% (w/v) sodium azide
  • the resulting anticoagulant- treated endotoxin sorbent may be incorporated in suitable plasmapheresis apparatus, and the present invention also provides an endotoxin sorbent according to the present invention in a form suitable for use in plasmapheresis apparatus, for example, in a chromatography column, and the present invention further provides plasmapheresis apparatus including an anticoagulant-treated polymyxin B-carrying solid phase support according to the present invention.
  • Surgical techniques for the insertion into a body of lines suitable for the removal and return of blood to be treated by plasmapheresis are known.
  • the blood removed is generally pumped through a plasma separator within which the plasma is separated from the cellular components of the blood.
  • the separated plasma is discarded, and the cellular components of the blood are returned to the body with replacement plasma or plasma substitute.
  • the separated plasma is passed through sorbent means comprising a sorbent material to adsorb or absorb certain materials either ⁇ on- specifically or specifically.
  • the treated plasma is then reunited with the cellular blood components and returned to the body.
  • the sorbent material may be a non ⁇ specific sorbent material, for example, powdered charcoal, or may comprise material suitable for affinity chromatography.
  • the sorbent material is conventionally present in the form of a column.
  • anticoagulant-containing solution for example, heparin-containing saline, for example, saline containing 10 units of heparin per ml, or donor plasma containing 10 units of heparin per ml.
  • the present invention provides a method of removing endotoxin from endotoxin-containing blood, which comprises separating plasma from the cellular components of the blood, and treating the plasma with an endotoxin sorbent according to the present invention.
  • the separation of the plasma and its subsequent treatment with the endotoxin sorbent may be carried out in the same apparatus or the separated plasma may be treated in a separate endotoxin sorbent-containing apparatus.
  • the method of removing e'ndotoxin from endotoxin- containing blood comprises, for example, subjecting the blood to plasmapheresis in apparatus comprising anticoagulant-treated, polymyxin B-bearing solid phase support according to the present invention as a sorbent means.
  • the second method of plasmapheresis described above may be used in the present invention, that is to say, the method whereby the separated plasma is treated with sorbent means, reunited with the cellular components and returned to the body.
  • separated plasma is treated "on line" with the sorbent means.
  • plasma may be separated from endotoxin-containing whole blood in a plasma separator and then treated on separate apparatus, for example, a column comprising an endotoxin sorbent according to the invention. The treated plasma may then if desired, be reunited with the cellular components of the blood, before return to the body.
  • the method of the present invention includes both of these embodiments.
  • the sorbent means may be any of the forms of anticoagulant-treated, polymyxin B-bearing solid phase support described above.
  • the sorbent means is preferably in the form of a column, and the solid support is especially agarose or polystyrene.
  • the anticoagulant is especially heparin.
  • the sorbent means may be pre-treated with the same anticoagulant as is used to prime the extra-corporeal circuit, or a different anticoagulant may be used. It is preferable to use the same agent both for pre- treatment of the solid support and for priming the extra-corporeal circuit. Heparin is preferably used.
  • the sorbent means incorporated in the plasmapheresis apparatus may be polymyxin B-bearing solid phase support that is treated _in situ with anticoagulant.
  • the amount of anticoagulant to be circulated through the solid component is indicated above, and should be an excess, especially a large excess compared with the amount of anticoagulant used conventionally for priming plasmapheresis apparatus, for example, in the case of heparin with agarose beads (in the form of Sepharose 4B) , amounts of heparin in excess of 100 units per ml agarose gel are recommended, preferably 500 units or more, and especially 1,000 units or more, for example, 5,000 units or more, for example, 10,000 units.
  • the endotoxin-rem ⁇ val component of the plasmapheresis apparatus may be used in conjunction with one or more other components suitable for removing substances from plasma, either specifically or non- specifically, that is to say, one or more other sorbent means. These other components may be used in series or in parallel with the endotoxin-removing component.
  • components for removing other substances from plasma are activated charcoal, broad- based sorbents used in the treatment of acute poisoning, chronic and acute renal failure; particles suitable for the removal of non-polar solutes from polar media for example, plasma; non-ionic acroreticular resins; sorbents with a particular attraction for lipid-soluble molecules; anionic exchange resins; sorbents suitable for the removal of protein-bound solutes, for example, bound antibodies having a selective removal action; artificial cells; and immobilised or encapsulated enzymes.
  • the present invention includes such mixed sorbents.
  • endotoxin may be removed from the sorbent using certain buffer systems, for example, a solution containing deoxycholate, for example, 1% (w/v) deoxycholate, thus regenerating the sorbent.
  • a solution containing deoxycholate for example, 1% (w/v) deoxycholate
  • the efficacy of the anticoagulant-treated polymyxin B bearing solid phase support as sorbent in the removal of endotoxin can be tested _in_ vitro.
  • assays which can be used for determining endotoxin levels, for example, those using imulus amoebocyte lysate.
  • this assay see for example Cohen and McConnell (J. Infect.
  • Polymyxin B may be assayed in a conventional manner, for example, using the standard bioassay which employs Bordetella bronchiseptica ATCC 4617 (see Sullman S.C. Polymyxins In: Reeves D.S., Philips I., Williams J.D. and Wise R. , Laboratory methods in antimicrobial chemotherapy, 1st Ed. Churchill- Livingstone, 1978, 232-234).
  • the assay methods described above may also be used in connection with plasmapheresis.
  • polymyxin B remains substantially immobilised on the solid phase support and is not eluted off during treatment of plasma. This is an important advantage, as* polymyxin B is neurotoxic and nephrotoxic.
  • the present invention also provides endotoxin sorbent immobilised on a solid phase support, the solid phase-endotoxin sorbent having been treated with an anticoagulant capable of preventing or limiting fibrin deposition, whereby free anticoagulant binding sites are blocked, apparatus comprising the sorbent and a method of removing endtoxin from blood using the sorbent.
  • an anticoagulant capable of preventing or limiting fibrin deposition, whereby free anticoagulant binding sites are blocked
  • apparatus comprising the sorbent and a method of removing endtoxin from blood using the sorbent.
  • the tube containing the Sepharose was then sealed and incubated at 4°C, for 18 hours on a mechanical rotator. Next, the gel was centrifuged at 80 0 g and excess buffer removed. Ten ml of 1 M ethanolamine pH7 were added and the tube incubated for 2 hours at room temperature. After further gentle centrifugation the supernatant was removed and the gel washed in three alternating cycles of acetate buffer (0.1 M sodium 5 acetate with 0.5 M NaCl final pH 4.0) and borate buffer (0.1 M sodium tetraborate with 0.5 M NaCl final pH 8.0).
  • acetate buffer 0.1 M sodium 5 acetate with 0.5 M NaCl final pH 4.0
  • borate buffer 0.1 M sodium tetraborate with 0.5 M NaCl final pH 8.0
  • the gel was incubated on a rotary mixer for 1.5 hours at room temperature with heparinized sterile 0.9% saline using 10,000 units of 0 heparin per ml of gel.
  • the treated gel was washed with 0.9% saline and was stored at 4°C in 0.9% saline containing 0.02% sodium azide.
  • PB-Sepharose or antibiotic-free control Sepharose: CON-Sepharose
  • PB-Sepharose antibiotic-free control Sepharose
  • CON-Sepharose antibiotic-free control Sepharose
  • Plasmapheresis apparatus is described, by way of example only, with reference to Figure 1, which is a diagrammatic representation.
  • a rat 1 to which indwelling carotid arterial and jugular venous lines . 2 and 3 respectively had been inserted was kept in a container 4 and given food and water ad_ libitum .
  • the arterial blood is pumped, by a pump 5, through a specially designed plasma separator 6 into a reservoir 7.
  • the filtered plasma is pumped by a pump 8 through a column 9 containing the sorbent material.
  • the treated plasma is then reunited with the cellular blood components in a mixing means 10 and returned, pumped by pump 11, to the animal via jugular vein lines.
  • trans-membrane pressure can be maintained at less than 50 ⁇ ⁇ ⁇ Hg with a filtration rate of 0.2-0.22 ml/min.
  • one ' complete plasma volume (approximately 11-12 ml) can be treated in 50-60 minutes.
  • the extracorporeal circuit is primed with approximately 3 ml of heparinized saline, obviating the need for donor blood or plasma.
  • Previous studies in 15 normal animals have established that the system can be used repeatedly with no apparent side effects, nor marked effects on red cell, leucocyte or platelet counts.. A sieving coefficient of >98% for the plasma membrane was obtained for C3, IgC and albumen.
  • Plasma from four PB-Sepharose perfused animals was used to determine if PB was eluted from the column in vivo . Samples obtained at 5, 10 and 15 minutes and at 90, 120 and 240 minutes were pooled and assayed for PB as described.
  • Plasma endotoxin levels measured 'pre' and 'post' the PB-Sepharose and CON-Sepharose columns after injection of 10mg/g endotoxin followed by plasmapheresis are shown in Figure 3.
  • the peak level reached at 20 minutes was 260 ng/ml.
  • the 'pre' and 'post' column endotoxin levels were the same, indicating that there was no significant endotoxin clearance across the CON-Sepharose column.
  • the peak 'post' column endotoxin concentration was 13 ng/ml, and for most of the re ainder of the experiment was undetectable. It is of interest that endotoxin was present continually in 'pre' column samples from PB-Sepharose treated animals, despite the fact that endotoxin was undetectable in 'post' column samples for most of the experiment. Thus, the absorbent was effectively removing the endotoxin presented to it, but it appeared that further endotoxin (albeit at a low level) was being generated from the animal. The most plausible explanation is that this 'fresh' endotoxin is derived from the intestinal microflora.
  • Figure 5 illustrates the effect of the procedure on the platelet count. Animals perfused over the PB- Sepharose were protected substantially from thrombocyt ⁇ penia: the initial count was 550 + 37 x

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Abstract

Absorbant d'endotoxines comprenant une substance absorbante sélectionnée dans le groupe composé de polymyxine B et de ses sels, immobilisée sur un support en phase solide, l'absorbant phase solide-polymyxine B ayant été traité avec un anticoagulant pouvant empêcher ou limiter le dépôt de fibrine, les sites de fixation d'anticoagulant libre étant bloqués. Cette substance peut être utilisée pour extraire les endotoxines de fluides contenant des endotoxines, par exemple le sang dans le traitement du choc par des bactéries gram-négatives.
PCT/GB1987/000406 1986-06-13 1987-06-11 Elimination d'endotoxines Ceased WO1987007531A1 (fr)

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GB8614468 1986-06-13
GB08614468A GB2192633A (en) 1986-06-13 1986-06-13 Endotoxin removal

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WO1987007531A1 true WO1987007531A1 (fr) 1987-12-17

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WO (1) WO1987007531A1 (fr)

Cited By (8)

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EP0328256A1 (fr) * 1988-01-21 1989-08-16 Owens-Corning Fiberglas Corporation Fibres de verre recouvertes d'agarose servant de garnissage ou de milieu chromatographique pour colonne dans les bioséparations
WO1989009627A1 (fr) * 1988-04-07 1989-10-19 Edward Shanbrom Catheter intravasculaire non-trombogenique recouvert de polymixine
DE10045343B4 (de) 2000-09-14 2005-05-12 Neuhann, Tobias, Dr.med. Verwendung einer wässrigen Lösung oder Suspension zum Heilen des Hornhautepithels
US7799347B2 (en) * 2006-09-07 2010-09-21 Chondrex Inc. Endotoxin-adsorbent for the prevention and treatment of autoimmune diseases
EP2409995A2 (fr) 2007-02-28 2012-01-25 Lipoxen Technologies Limited Réduction des endotoxines dans les acides polysialiques
DE102015002073A1 (de) * 2015-02-18 2016-08-18 Fresenius Medical Care Deutschland Gmbh Vorrichtung zur Durchführung einer Apheresebehandlung
CN112552500A (zh) * 2021-01-04 2021-03-26 珠海麦得发生物科技股份有限公司 一种去除发酵法PHAs中内毒素的方法
US20210138143A1 (en) * 2013-12-27 2021-05-13 Eliaz Therapeutics, Inc. Plasmapheresis device

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CA2490467C (fr) 2002-06-24 2011-06-07 Profos Ag Methode de detection et d'elimination d'endotoxines
US6803183B2 (en) 2002-07-18 2004-10-12 Clarigen, Inc. Method for removing pyrogens from plasma and blood for treatment of septic shock
DE102005002969A1 (de) 2005-01-21 2006-08-03 Profos Ag Verfahren zum Nachweis und zur Entfernung von Endotoxin
EP2961760B1 (fr) 2013-02-26 2021-03-24 EMD Millipore Corporation Extraction sélective d'une protéine contenue dans un mélange de protéines au moyen de charbon actif par ajustement des conditions de la solution.
CN116510703B (zh) * 2023-04-07 2025-03-14 北京中科太康科技有限公司 一种提高抗凝血效果的吸附剂及其制备方法和应用

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0328256A1 (fr) * 1988-01-21 1989-08-16 Owens-Corning Fiberglas Corporation Fibres de verre recouvertes d'agarose servant de garnissage ou de milieu chromatographique pour colonne dans les bioséparations
WO1989009627A1 (fr) * 1988-04-07 1989-10-19 Edward Shanbrom Catheter intravasculaire non-trombogenique recouvert de polymixine
DE10045343B4 (de) 2000-09-14 2005-05-12 Neuhann, Tobias, Dr.med. Verwendung einer wässrigen Lösung oder Suspension zum Heilen des Hornhautepithels
US8287911B2 (en) 2006-09-07 2012-10-16 Chondrex Inc. Endotoxin adsorbent for the prevention and treatment of autoimmune diseases
US7799347B2 (en) * 2006-09-07 2010-09-21 Chondrex Inc. Endotoxin-adsorbent for the prevention and treatment of autoimmune diseases
EP2409995A2 (fr) 2007-02-28 2012-01-25 Lipoxen Technologies Limited Réduction des endotoxines dans les acides polysialiques
EP2409994A2 (fr) 2007-02-28 2012-01-25 Lipoxen Technologies Limited Réduction des endotoxines dans les acides polysialiques
US9212232B2 (en) 2007-02-28 2015-12-15 Lipoxen Technologies Limited Reduction of endotoxin in polysialic acids
US20210138143A1 (en) * 2013-12-27 2021-05-13 Eliaz Therapeutics, Inc. Plasmapheresis device
DE102015002073A1 (de) * 2015-02-18 2016-08-18 Fresenius Medical Care Deutschland Gmbh Vorrichtung zur Durchführung einer Apheresebehandlung
US10780211B2 (en) 2015-02-18 2020-09-22 Fresenius Medical Care Deutschland Gmbh Apparatus for carrying out an apheresis treatment
CN112552500A (zh) * 2021-01-04 2021-03-26 珠海麦得发生物科技股份有限公司 一种去除发酵法PHAs中内毒素的方法
CN112552500B (zh) * 2021-01-04 2022-12-20 珠海麦得发生物科技股份有限公司 一种去除发酵法PHAs中内毒素的方法

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GB8614468D0 (en) 1986-07-16

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