Classifications

• • 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
  • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
  • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
• • 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
  • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
  • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
  • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • 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
  • A61M2202/00—Special media to be introduced, removed or treated
  • A61M2202/04—Liquids
  • A61M2202/0413—Blood
  • A61M2202/0456—Lipoprotein

Definitions

• THIS INVENTION relates to plasma or serum delipidation in animals (which term shall indicate humans), to a treatment for cardiovascular disease and to removal of excess fat from the animals. In particular, it is directed to the removal of cholesterol, triglycerides and other lipids, and fat soluble toxins—for example, insecticides—from the blood plasma or serum of such animals.
• Cardiovascular diseases are responsible for a significant number of deaths in most industrialised countries.
• Atherosclerosis is characterised by local fatty thickening in the inner aspects of large vessels supplying blood to the heart, brain and other vital organs. These lesions obstruct the lumen of the vessel and result in ischaemia of the tissue supplied by the vessel. Prolonged or sudden ischaemia may result in a clinical heart attack or stroke from which the patient may or may not recover.
• diet is the basic element of all therapy for hyperlipidaemia (excessive amount of fat in plasma).
• hyperlipidaemia excessive amount of fat in plasma.
• use of diet as a primary mode of therapy requires a major effort on the part of physicians, nutritionists, dieticians and other health professionals.
• a combination of both drug and diet may be required to reduce the concentration of plasma lipids.
• Hypolipidaemic drugs are therefore used as a supplement to dietary control.
• Plasmapheresis (plasma exchange) therapy has been developed and involves replacement of the patient's plasma with donor plasma or more usually a plasma protein fraction. This treatment can result in complications due to the possible introduction of foreign proteins and transmission of infectious diseases. Further, plasma exchange removes all the plasma proteins as well as very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL).
• VLDL very low density lipoprotein
• LDL low density lipoprotein
• HDL high density lipoprotein
• HDL is inversely correlated with the severity of coronary arterial lesions as well as with the likelihood that these will progress. Therefore, removal of HDL is not advantageous.
• aphaeresis techniques also exist which can remove LDL from plasma. These techniques include absorption of LDL in heparinagarose beads (affinity chromatography) or the use of immobilised LDL-antibodies. Other methods presently available for the removal of LDL involve cascade filtration absorption to immobilised dextran sulphate and LDL precipitation at low pH in the presence of heparin. Each method specifically removes LDL but not HDL.
• LDL aphaeresis has, however, disadvantages. Significant amounts of other plasma proteins are removed during aphaeresis and to obtain a sustained reduction in LDL-cholesterol, LDL aphaeresis must be performed frequently (up to once weekly). Furthermore, LDL removal may be counter productive as, low blood LDL levels may result in increased cellular cholesterol synthesis.
• cholesterol aphaeresis an extra corporeal lipid elimination process, termed “cholesterol aphaeresis”, has been developed.
• cholesterol aphaeresis blood is withdrawn from a subject, plasma separated from the blood and mixed with a solvent mixture which extracts lipid from the plasma, after which the delipidated plasma is recombined with the blood cells and returned to the subject.
• cholesterol aphaeresis results in the removal of fats from plasma or serum.
• the proteins that transport the fat remain soluble in the treated plasma or serum.
• the apolipoproteins of VLDL, LDL and HDL are present in the treated plasma or serum.
• These apolipoproteins in particular apolipoproteins A1 from the defatted HDL in the plasma or serum, are responsible for the mobilisation of excessive amounts of deposited fats such as cholesterol in arteries, plaques, or excessive amounts of triglycerides, adipose tissue, or fat soluble toxins that are present in adipose tissue.
• These excessive amount of fats or toxins are transferred to the plasma or serum, bound to the newly assembled lipoproteins.
• these unwanted fats or toxins are successively removed from the plasma and thus the body.
• cholesterol aphaeresis has overcome the shortcomings of dietary and/or drug treatments and other aphaeretic techniques
• existing apparatus for cholesterol aphaeresis does not provide a sufficiently rapid and safe process.
• apparatus is required which effects delipidation more efficiently.
• flow rates of the order of 70 ml/min are required for cholesterol aphaeresis of a human subject.
• the first disadvantage is the explosive nature of the solvents used to delipidate this plasma. These solvents are, by the very nature of the continuous systems, in close proximity to the patient and medical staff. This hazard is clearly present for the duration of the delipidation process which usually runs for several hours.
• the second disadvantage is that, in the prior continuous systems, a reliable procedure is not available to remove totally all of the solvents used in the delipidation before the treated plasma is returned to the patient.
• the use of the preferred solvent 1-butanol in the delipidation is of concern as it can now be established that that solvent can be present as 1% to 5% of the treated plasma that is returned to the patient.
• a third disadvantage is that the continuous systems described above are not suitable for the delipidation of serum. If serum can be delipidated, there would be the advantage of favourably altering the blood rheology in that the viscosity will decrease following delipidation resulting in better haemodynamics for the originally impaired blood circulation.
• a fourth disadvantage is that delipidation in a continuous system is undertaken over several hours. Apart from the prolonged exposure to the hazardous solvents as discussed above, the equipment and staff are committed to a single patient. As the removal of plasma or other blood fractions and their subsequent return to the patient as individual steps each only take a few minutes, it would be advantageous if the relatively lengthy delipidation step could be undertaken off site, thus freeing the patient, medical staff and equipment for other matters.
• a method for the removal of cholesterol, triglycerides and other lipids from animal plasma, serum or other suitable blood fractions comprising withdrawing blood from a subject, separating the required fraction from the blood and mixing with a solvent mixture which extracts the said lipids from the fraction, after which the delipidated fraction is recombined with the blood cells and returned to the subject, characterised in that the solvent extraction step is carried out separately and remote from the subject.
• beads are used when mixing the blood fractions with the solvent. More preferably, the beads have a density substantially mid-way between the density of the fraction and the density of the solvent mixture. This ensures efficient mixing with a large surface area, increasing the efficiency of the extraction and also serving as a good separator of the plasma from the solvent when centrifugation is used to isolate the phases after extraction.
• the beads Preferably, to obtain a density substantially mid-way between the density of the fraction and the density of the solvent mixture, the beads contain entrapped air.
• the density of plasma is approximately 1.006 g/ml and the solvents used generally have a density of approximately 0.8 g/ml, the density of the beads will be around 0.9 g/ml.
• the beads may be manufactured from any acceptable material such as glass or plastic.
• One way of removing this solvent is to wash with another solvent, preferably diethyl ether, to remove substantially all of the original solvent used in the extraction step.
• efficient removal of the extraction solvent can be achieved by mixing the delipidated fraction with an absorbent specific for the solvent that is being removed.
• the absorbent is contained in the pores of sintered spheres.
• the sintered spheres are approximately 2 to 5 mm in diameter with the pores of the spheres being less than 50 ⁇ in diameter.
• the spheres are manufactured from glass.
• the absorbents used in the sintered spheres are the macroporous polymeric beads for absorbing organic molecules from aqueous solutions marketed by Bio-Rad Laboratories under the trade name Bio-Beads SM.
• the absorbent is preferably Bio-Beads SM-2.
• the absorbent is held in a chamber which is adapted to allow the delipidated fraction to pass through or over the absorbent at least twice if a single pass is insufficient to remove all of the solvent.
• phase is subsequently washed with another solvent, preferably diethyl ether, to remove a substantial amount of the original solvent before the treatment with the absorbent.
• another solvent preferably diethyl ether
• phase is washed at least three (3) times.
• the plasma may be human plasma or plasma from other living animals.
• the plasma can be obtained from human or animal blood by known plasma separating techniques which include centrifugal separation, filtration and the like.
• the serum or other lipid-containing fraction can be derived from human or other living animals by known techniques.
• Suitable solvents for the extraction comprise mixtures of hydrocarbons, ethers and alcohols.
• Preferred solvents are mixtures of lower alcohols with lower ethers.
• the lower alcohols suitably include those which are not appreciably miscible with the plasma and these can include the butanols (butan-1-ol and butan-2-ol).
• C 1-4 ethers are also preferred and these can include the propyl ethers (di-isopropyl ether and propyl ether).
• Other solvents which may be applicable include amines, esters, hydrocarbons and mixtures providing that the solvent can (1) rapidly and preferably remove cholesterol from the plasma, (2) is substantially immiscible with the plasma, (3) can be removed from the plasma, and (4) does not denature the desired moieties.
• Preferred solvent compositions are butanol with di-isopropyl ether and these may be in the ratio of 0%-40% of the alcohol to 100%-60% of the ether.
• the roosters used in this study were of White Leghorn Hiline strain and were obtained as one-day old chicks. All roosters from 8 weeks old were transferred into individual cages. Water and feed were supplied unrestricted. At eight weeks of age, 15 control birds were fed a commercial poultry ration for 31 days and another group of 30 birds were injected subcutaneously each day with 5 mg diethylstilboestrol (DES) in sesame oil for a period of 31 days. In addition they were fed on the same commercial diet which was supplemented with 2.6% (w/w) cholesterol for a period of 31 days. Fifteen animals of the DES treated group were then subjected to lipid aphaeresis (LA).
• LA lipid aphaeresis
• Approximately 25% of the calculated blood volume was collected from a brachial vein of the animal with a 21 gauge needle and syringe. The total blood volume was estimated at 8 percent of the body weight.
• the blood was collected in heparinized tubes and immediately centrifuged at 900 g for 5 minutes at room temperature. The blood cells were suspended in an amount of saline equivalent to the plasma volume and were reinfused into the animal.
• the plasma was kept refrigerated for twelve hours and was then delipidated for 20 minutes with a mixture of butanol and di-isopropyl ether (DIPE), 25:75 (v/v), in a ratio of one volume of plasma to two volumes of butanol-DIPE mixture (organic phase).
• DIPE butanol and di-isopropyl ether
• Inert plastic beads with a density of 0.9 g/mL (1 g) were added to the mixture. After extraction, the mixture was centrifuged at 900 g for 2 min to separate the plasma and organic phases. The organic phase (upper layer) was removed, free of plasma phase, by careful aspiration with a pasteur pipette under vacuum. Traces of butanol in the plasma phase were washed out with four volumes of diethyl ether (DEE) for 2 min by end-over-end rotation at 30 rpm. The mixture was then centrifuged at 900 g for 2 min to separate plasma and ether phases. The ether phase was subsequently removed by aspiration with a pasteur pipette. Residual ether was removed by evacuation with a water pump aspirator at 37° C. The plasma was then passed through a 5 mL column containing Bio-Beads SM-2.
• DEE diethyl ether
• This procedure yielded delipidated plasma.
• the delipidated plasma was re-mixed with the blood cells of a subsequent 25% blood collection which was then reinfused through a brachial vein back into the identical donor animals.
• the duration of the entire procedure that is, removal of blood from the animal to reinfusion of treated blood back to the animal was approximately 1 hour.
• the animals were sacrificed and their livers and aortae were dissected.
• the LA treatment procedures were repeated 3 times after the first treatment.
• the blood was collected in heparinized tubes and immediately centrifuged at 900 g for 5 min.
• the plasma was separated from the blood cells.
• the blood cells were mixed with saline in the same volume of the collected plasma and reinfused into the animal.
• the plasma was kept refrigerated for twelve hours and was then remixed with blood cells of a subsequent 25% blood collection after the second and/or subsequent plasma separations.
• the animals were sacrificed and their livers and aortae were dissected.
• the sham treatment procedures were repeated 3 times after the first treatment.
• the livers were weighed, minced with a scalpel blade and homogenised in 0.9% sodium chloride solution by 10-12 strokes of a motor driven Teflon-glass homogeniser (1900 rpm).
• the aorta was weighed and three times its weight of 3 mm glass beads were added in a homogenising bottle containing 0.9% sodium chloride. The contents were then homogenised for one minute.
• the lipid from the homogenised liver and aorta samples were extracted by the Folch procedure and weighed.
• Plasmapheresis is performed using vein-to-vein or arteriovenous fistula in the forearm of patients. Heparin is given at the beginning of the procedure as a 5,000 unit bolus, and then by continuous infusion at the rate of 700 units per hour over the course of the procedure. Access through the antecubital veins should provide plasma flow rates of 25 to 40 mls per minute.
• ACD-A anticoagulant
• the plasma is kept refrigerated up until twelve hours prior to reinfusion of delipidated plasma in exchange for another twenty five percent plasma collection (weekly or biweekly).
• the plasma is delipidated and the delipidated plasma is tested to ensure all solvent has been removed before the clean delipidated plasma is exchanged for new untreated plasma.
• the continuous flow system described in U.S. Pat. No. 4,895,558 (the entire content of which is included herein) is modified to a discontinuous system by removing the appropriate blood volume to be treated and subjecting that volume to delipidation at a site remote from the patient.
• the continuous flow system described in International Patent Application No. PCT/AU94/00415 (the entire content of which is included herein) is modified to a discontinuous system by removing the appropriate blood volume to be a site remote from the patient before the plasma is dispersed into small droplets into the solvent by the dispersing means.
• the extraction step can include, in accordance with the present invention, either multiple washing of the extracted phase and/or using an absorbent.
• the plasma is delipidated with a solvent mixture comprising 1-butanol and di-isopropyl ether.
• the delipidated fraction is then washed three (3) or four (4) times with diethyl ether.
• the diethyl ether is removed by centrifugation and vacuum extraction at 37° C.
• the sintered spheres containing Bio-Beads SM-2 are then mixed with the delipidated plasma to remove the final traces of 1-butanol.
• Discontinuous LA treatments corresponding to approximately one plasma volume treated by four applications of 25% of plasma volume treated per time resulted in significant decreases in both hepatic and aortic lipids in hyperlipidaemic animals.
• the LA treated hyperlipidaemic animals ended up with lipid values that were similar to control animals.
• the present invention can remove or at least significantly reduce any danger to patients and medical staff from the explosive nature of the solvents employed.
• the improved solvent extraction method of the present invention is not limited to plasma delipidation but also it is applicable to the delipidation of serum, thus providing advantageous changes to the blood rheology of the originally impaired blood circulation of the patient.
• the present invention thus provides for a rapid regression of coronary atherosclerosis in a patient.
• the present invention is a discontinuous system, it is not essential to return the delipidated blood fraction immediately to the patient. It is already known that plasma or serum can be collected and stored under sterile conditions in a refrigerator or freezer for extended periods and that it can be returned safely to the patient within twelve (12) hours of breaking the sterile seal. Therefore, if necessary, reintroduction of the delipidated fraction can occur several weeks after it was first removed from the patient. This option leads to particular advantages such as, economies of scale when several patients have to be treated simultaneously, the freeing of medical staff and equipment for other duties, and the reduction in stress for the patient whom no longer has to be hooked up to a delipidation apparatus for several continuous hours.

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Citations (104)

• 1994
  • 1994-12-22 AU AUPN0307A patent/AUPN030794A0/en not_active Abandoned
• 1995
  • 1995-12-22 CA CA002208059A patent/CA2208059A1/fr not_active Abandoned
  • 1995-12-22 JP JP8519368A patent/JPH10510739A/ja active Pending
  • 1995-12-22 EP EP95941995A patent/EP0799061B1/fr not_active Expired - Lifetime
  • 1995-12-22 AU AU43236/96A patent/AU695826B2/en not_active Ceased
  • 1995-12-22 WO PCT/AU1995/000875 patent/WO1996019250A1/fr not_active Ceased
  • 1995-12-22 DK DK95941995T patent/DK0799061T3/da active
  • 1995-12-22 PT PT95941995T patent/PT799061E/pt unknown
  • 1995-12-22 US US09/879,002 patent/USRE39498E1/en not_active Expired - Fee Related
  • 1995-12-22 AT AT95941995T patent/ATE355864T1/de not_active IP Right Cessation
  • 1995-12-22 ES ES95941995T patent/ES2282995T3/es not_active Expired - Lifetime
  • 1995-12-22 DE DE69535418T patent/DE69535418T2/de not_active Expired - Lifetime
• 2005
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