AU594964B2 - Autologous plasma delipidation using a continuous flow system - Google Patents

Description

5949 64 Tlit d0ocnUr t coamlat Lb* .0n0a1teets ade 8w Sec i 49.

*nd is a hr p<tofl«i I COMMONWEALTH OF AUSTRALIA The Patents Act 1952

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Name of Applicant Address of Applicant Actual Inventor Address for Service UNIVERSITY OF QUEENSLAND St. Lucia, Queensland, 4067, Australia.

BILL ELLIOT CHAM GRANT ADAMS COMPANY, Patent Trade Mark Attorneys, 333 Adelaide Street, BRISBANE, QUEENSLAND, 4000

AUSTRALIA.

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04 4 COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: ,,AUTOLOGOUS_ P LM mPIDATION USING A CONTINUOUS FLOW SYSTEM" The following statement is a full description of the invention including the best method of performing it known to us: I :I THIS INVENTION relates to a method of and apparatus for autologous plasma delipidation in animals (which term shall include humans) using a continuous flow system.

Cardiovascular diseases are responsible for more than half of all deaths in Australia and tKe United States and are frequent in most industrialized countries. At present, an Australian or American boy has a one in five risk of developing clinical evidence of coronary heart disease (CHD) before his 65th birthday.

Atherosclerosis is characterised by focal 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.

It is believed that atherosclerosis begins in early childhood evolving through various stages before ,becoming clinically apparent in middle and late adult o life. The earliest grossly recognizable intimal lesion is the fatty streak composed by lipid material, connective tissue and other substances. It is present in the aorta of many children less than 3 years of age. Fatty streaks become evident in the coronary arteries during the second decade, being seen in nearly all cases of CHD 0 after 20 years of age. The frequency of coronary fatty streaks parallels the development of CHD. The most striking tiochemical abnormality in human atherosclerosis is the accumulation of massive amounts of cholesteryl esters in the core of the atheromatous plaque.

The relationships among dietary lipid, serum cholesterol and atherosclerosis have long been recognised.

In many epidemiological studies it has been shown that a 1- _i I i single measurement of serum cholesterol has proved to be a significant predictor of the occurrence of

CHD.

There seems little dcebt concerning the relationship between elevations of plasma cholesterol and the development of premature CHD in humans. The fraction of cholesterol carried in low-density lipoproteins (LDL) appears to be particularly atherogenic.

Thus, elevations of LDL and LDL-cholesterol apparently predispose individuals to an accelerated form of atherosclerosis.

As an individual grows larger and older, there is a net accumulation of cholesterol in body tissues, including the arterial wall. The body can rid itself o 15 of substantial quantities of cholesterol only through o* the liver, where cholesterol can be excreted in the bile and in the faeces. A mechanism has not yet been firmly established to explain how cholesterol from *o peripheral tissues, including the arterial wall, is transported to the liver for removal. It has been suggested that high-density lipoprotein (HDL) plays a role in removing cholesterol from tissues and promoting o, 0 reverse cholesterol transport to the liver.

Diet is the basic eleme -t of all therapy for 0.

o 25 hyperlipidaemia (excessive amount of fat in plasma).

The combination of both drug and diet may be required to reduce the concentrations of plasma lipids. Hypoo S. lipidaemic drugs are therefore used as a supplement to dietary control. Many are effective in reducing blood lipids, but no drug works in all types of hyperlipoproteinemia and all have side-effects. There is no conclusive evidence that hypolipidaemic drugs can cause regression of atherosclerosis.

Over the past years the inventor and co-workers have focussed their attention on the development of a -3-

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o 0 o 9 o 0U o *44 *s 9 biphasic solvent system for extraction of biological solutions which would meet the following requirement: delipidation should be completed in a short period of time; (ii) the treatment should be mild enough so as not to cause irreversible denaturation of proteins, including enzymes; (iii) the resulting apolipoproteins(defatting lipoproteins) should remain soluble in the aqueous phase; (iv) the apolipoproteins should be able to dissolve lipids; recovery of the aqueous phase after extraction should be high; and 15 (vi) the method should be flexible i.e. applicable to small and large volumes of biological solutions as required.

Using an appropriate in vitro solvent system reported in a number of scientific papers, the inventor and his co-workers have shown that: del-pidation can be achieved in a matter of minutes; (ii) the procedure does not effect enzyme activities, proteins and ionic constituents; (iii) the apolipoproteins remain soluble; (iv) the apolipoproteins are able to bind and dissolve lipids (especially the HDL fraction); the recovery of the aqueous phase after extraction is very high; (vi) the procedure is very flexible.

A hyperlipaemic plasma looks turbid before delipidation. After delipidation, the turbid plasma becones clear because the lipids are extracted from the plasma into the organic phase. The amount of lipid extracted from plasma can be measured after evaporation of the organic phase, ~-"sriEflffrrr~ra~ While the systems discussed above have been successful in vitro in the experimental scale, they have not been suitable for the clinical delipidation of animal plasma.

The present invention is directed to the upgrading of the experimental in vitro systems to a clinical system. As a consequence, a number of preferred objects of the present invention include: 1. To apply a delipidation (defatting) procedure, developed by the invention, to prevent and reverse atherosclerosis (fatty thickening in the inner aspects of large vessels supplying blood to the heart, brain and other vital organs) to animals, including humans 1 2. That such a procedure is non-toxic; 3. To ensure that other substances such as fatsoluble vitamins which are removed by the defatting procedure are reintroduced to the subject; 4. To develop a machine that will be able to defat blood plasma on a continuous mode.

In one aspect the present invention resides in a method for autologous plasma delipidation of animals e i using a continuous flow system including: t drawing blood from the animal; separating the plasma from the red blood cells; 25 delipidating the plasma with a lipid solvent; remixing the delipidated plasma with the red Sblood cells; and re-introducing the delipidated blood into the I animal.

S1 30 Preferably the delipidation step includes the steps of: mixing the plasma with the liquid solvent; allowing the mixture to separate into an organic solvent/lipid chase and an aqueous delipidated plasma phase; and i I drawing off the organic phase.

In a second aspect, the present invention resides in an apparatus for autologous plasma delipidation of animals including: means to draw blood from an animal; means to separate the plasma from the red blood cells; means to delipidate the plasma using a lipid solvent; means to separate the delipidated plasma from the solvent; means to remix the delipidated plasma with the red blood cells; and means to re-introduce the delipidated blood into the animal.

The apparatus may be based on a double centrifuge blood cell separation machine. The blood may be drawn from the animal via a drawing needle and the blood may be mixed with a prime solution (and anticoagulant) and fed to a first centifugal separator where the blood is <separated into the plasma and red blood cells. The plasma may be fed to container of solvent or the plasma and solvent may be mixed in continuous flow lines. After mixing the plasma and the solvent, the mixture is preferably passed through a primary separator where the denser delipidated plasma (in an aqueous phase) is drawn off oI below the solvent/lipid components( in an organic phase).

Preferably the plasma is passed through a second centrifugal separator to remove any remaining traces of solvent before the delipidated plasma is remixed with the red blood cells and re-introduced to the animal e.g.

by an re-infusion needle.

The lipids may be extracted from the solvents e.g.

by distillation of the solvent and the solvent recycled through the apparatus.

-6- The preferred solvent is DIPE (di-isopropul-ether) which is preferably peroxide free, which may be mixed with butanol, 1-pentanol and/or 1-hexanol in the range 25%:50% to The most preferred solvent is butanol DIPE 40%:60%(V/V).

TO enable the invention to be fully understood, preferred embodiments will now be described with reference to the accompanying drawings, in which: FIG. 1 shows the build-up of deposits in the aorta of human with age; FIG. 2 is a graph of the level of cholesterol with time in two dogs treated in accordance with the present invention; H FIGS. 3 and 4 are graphs of the mechanism which leads to the build up of cholesterol in the arteries; FIG. 5 is a graph of the mechanism which is t believed to occur by the present invention which removes the cholesterol from the arteries; and S t FIG. 6 is a schematic view of a machine suitable for the present invention.

"'he influence of plasma delipidation was studied l in two dogs (one normocholesterolaemic and the other hypercholesterolaemic).

Approximately one-third of the blood volumes of the dogs were removed. The red blood cells were removed from the plasma by centrifugation. The plasma was delipidated, then remixed with the red blood cells and re-introduced back into the original dog by intravenous infusion. Blood samples were collected before and after this delipidation procedure for biochemical and haematological analyses.

During the first 15 minutes after the intravenous infusion of the autologous delipidated plasma, a low level of cholesterol was detected in the dogs, which gradually increased in 24 hours to approximately the -7i; :dr- L~~-i initial level in the normocholesterolaemic dog (FIG.

2, circles). However, in the hypercholesterolaemic dog the plasma cholesterol level did not return to ite initial level during the following eight days (FIG. 2, squares).

Twenty-four hours after reinfusion of the delipidated plasma, the plasma cholesterol conccntration in the initially hypercholesterolaemic and normocholesterolaemic dogs remained within the normal rane (3.24-6.48 mmol/1) and remained unchanged over the experimental period.

During the experimental period there wore no changes in the following biochemical and hacmatological parameters: 15 Biochemical Hacmatological Bilirubin W3C Total protein RBC Albumin Hacemalobin 00 r 0: 01+ 0 4; 0,r0 0 4 0004; 0 0 4 4; 0000 0 0 0 *rO 0 00* 0 0 C *0 a a 4 0 Total globulin 20 alpha 1 alpha 2 beta and gamma globulins Sodium Potassium Chloride Total Carbon dioxide Calcium Phosphate Urea Urate Creatinine Alkaline Phosphatase Lactate dehydrogenase Aspartate transaminase Creatine hinase Amylase Hot Mc

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Polymorph LymphocytoG Monocytes Eosinophils Platelets -8-

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Nucleotidase Gamma-glutamyl transpeptidase Anion gap Alpha 1 Antitrypsin From these very preliminary experiments it appears that delipidation of autologous plasma results in removal of lipids. The extracted plasma when reinfused into the animal picks up lipids, in particular, cholesterol, possibly from body stores. The procedure does not appear to have adverse effects on haematological and biochemical constituents. The animals appeared to be physically unaffected by the treatment.

In this connection using the butanol-DIPE solvent i S*T system, it has recently been shown that partial delipid- 15 ation of human LDL (removal of 98% cholesterol, 95% trioeltiu glyceride and 50% phospholipid) did not effect the binding activity of this treated LDL to cultured human fibroblasts when compared with untreated LDL. Moreover, delipidated LDL did not stimulate cholesteryl ester synthesis. On the 20 other hand, many investigators using the butanol-DIPE sysa tem, have subsequently shown that cellular sterol synthesis is highly stimulated by delipidated serum. More recently it has been shown that this stimulatory effect may be caused by delipidated HDL which is present in delipidated serum.

The induction is thought to be caused-by removal of cellular sterol by apo-HDL. When an acceptor of cheolesterol (apo- HDL) is present in the medium, macrophages excrete massive amounts of cholesterol. Substances in the delipidated d 1.215 g/ml fraction of plasma were shown to remove cholesterol from macrophages. Albumin and globulins did not accept large amounts of cholesterol. It was suggested that apolipoproteins (especially apo-A-I) may have been responsible for the activity of this fraction.

With respect to atherosclerosis the infusion of delipidated plasma may prove useful in an attempt to induce rapid endothelial repair by inhibition of the proliferat- -9-

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ive response. Partly delipidated plasma in the vascular system may inhibit cellular intake of cholesterol (by binding cholesterol-depleted LDL) and at the same time facilitate reverse cholesterol transport (incrsased cholesterol eiflux by reducing cellular sterol content with delipidated HDL). Lipoprotein lipases and lecithin cholesterol acyltransferase may also be activated by their apolipoprotein cofactors.

Atherosclerosis can also be defined as a disease process that occurs when the influx and deposition of cholesterol into the arterial wall exceeds the egress of cholesterol from the arterial wall. This process is 0 characterized by early deposition of cholesterol in 15 and around arterial wall cells. The cholesterol is derived from certain types of lipoproteins. While these lipoproteins deliver cholesterol to the arterial wall cells, other types of plasma lipoproteins may be capable of removing cholesterol from the cells and transporting the cholesterol to the liver from excretion ,0 from the body.

o* o Foam cells, which accumulate cholesterol in the arterial wall, are thought to originate from smooth S, cells and/or macrophage monocytes. Studies have demonstrated that cholesteryl ester accumulation in macrophages is caused by the uptake of diet-induced beta-VLDL, 00 and that the degree of this accumulation is modulated by the presence of HDL. It was suggested that the level 0 of lipoproteins capable of delivering cholesterol to macrophages beta-VLDL) versus the level of lipoproteins capable of acquiring cholesterol from the cells HDL) may determine whether or not atherosclerosis develops in the in vivo situation (see FIG. 3).

The preliminary studies in dogs have shown that it is possible to extract lipids, in particular cholesterol from plasma without effecting other plasma sub- 4 stances. This observation is of utmost importance and is the key to the clinical application. It has also been shown that the delipidated plasma (in particular apo-HDL) is capable of dissolving lipids including cholesterol. These phenomena may explain the observation that when autologous delipidated plasma is reinforced into an animal we first observe a decline in plasma cholesterol (because of the removal of cholesterol). It is quite feasible that the delipidated plasma (in particular apo-HDL as shown by in vitro studies) dissolves and binds cholesterol which had previously been deposited in tissues. This explanation is to a certain extent enforced by the in vivo dog studies (15 minutes after reinfusion of the delipidated f 15 plasma, the delipidated plasma gradually picks up cholesterol until the plasma level of cholesterol reaches its normal level). If this cholesterol is dissolved from atherosclerotic plaques one would expect regression of atherosclerosis to occur.

The occurrence of atherosclerosis progresses when the influx of cholesterol into the arterial wall exceeds the level of egress from this tissue (FIG. 4).

LDL and diet-induced beta-VLDL, which are cholesterci- 2° rich lipoproteins have been shown to deliver large amounts of cholesterol to macrophages in vitro and have been implicated in arterial disease in animals in vivo. The role of HDL in reverse cholesterol trans- 4 port from cells of the arterial wall to the liver with subsequent excretion through the bile into the intestine is also shown. The red crosses depict cholesterol carried by the lipoprotein fractions and the deposited cholesterol in the artery (FIG. FIG. 5 shows the possible effects of the removal, delipidation and reinfusion of plasma in a continuous mode in the body system. The red crosses depict cholesterol carried by -11- I the lipoprotein fractions and the deposited cholesterol in the artery. Delipidation of plasma results in reduction of cholesterol in the lipoprotein fractions. The cholesterol-depleted LDL and beta-VLDL can still bind to receptors. On the one hand, atherosclerosis will be partly or completely arrested because the influx of cholesterol into the arterial wall is reduced. On the other hand, the egress of cholesterol from the arterial plaque in increased by the cholesterol-depleted HDL.

Cholesterol-depleted HDL is capable of dissolving and binding cholesterol (see earlier). In this connection one would expect that as a consequence of plasma delipidation the cholesterol-depleted HDL will 15 dissolve cholesterol from the atherosclerotic plaque.

This cholesterol which is now HDL-bound is then taken to the liver (reverse cholesterol transport) which may then be excreted through the bile and intestine in the faeces. Thus reinfusion of autologous plasma results in a REDUCTION of INFLUX of cholesterol into the arterial wall and an INCREASE of EGRESS of cholesterol from o this tissue (regression of autherosclerosis).

Because we are using autologous plasma (the patient's own plasma is reintroduced) there will be no problems of rejection or antibody production.

Another beneficial effect of plasma delipidation is removal of adipose tissue (triglycerides) from the body. This procedure may also be applicable for detoxifying the body from fat-soluble toxins such as some herbicides and insecticides e.g. 2,4-D; 2,4,5T and the organo phosphates.

It may also be possible to use the present method to treat, or at least arrest, the spread of cancer.

The quickly-dividing cancer cells require lipids to produce the membranes for the newly generated cells.

-12i- By removing the lipids from the plasma, the necessary building blocks for the cancer cells may be removed and so the mature cancer cells may die without dividing. Even if the cells do divide, the absence (or low level) of the lipids will reduce the rate of division arresting or retarding the rate of the spread of the cancer cells.

Referring to FIG. 6, a schematic view of a machine suitable for the invention is shown based on the standard blood cell separation.

The blood from the animal is drawn via a drawing needle and is mixed with an anticoagulant solution, t drawn from a supply container via a metering pump.

f, The mixture passes through a vein monitor, drawn by a blood pump, and is fed to a first disposable centrifugal separator where the blood is separated into red S,,1 blood cells, plasma and waste products (the latter being e *collected in a waste bag). The red blood cells are drawn through a red cell pump to a level monitor to be mixed with the delipidated plasma, supplied by the replacement fluid pump, for return to the animal via the Sor o reinfusion needle.

The plasma is mixed with butanol-DIPE (40%:60% 4.

o V/V) solvent drawn from a supply via a solvent pump.

The plasma and solvent is mixed and the solvent removes the lipids from the plasma. The mixture is then passed o 00 othrough a plasma/solvent lipid combination (in the phase). In a simple laboratory experimental unit, the separation unit comprised a glass tubing unit with a Yshape with the arms of the Y vertically aligned. Most of the solvent and lipid, in the less dense organic phase, flowed out through upper arms while the plasma in the aqueous phase flowed out through the lower arms. Ether, which breaks down any emulsion in the plasma and which -13- I I -I I d removes any remaining solvent from the plasma is added after the separation unit. The plasma is then pumped through a second disposable centrifugal separator where the balance of the solvent and lipids (and the ether) (all in the organic phase) are removed and directed to a collector. (The solvent is distilled off from the lipid and the solvent is pumped back to the solvent supply tank in a recycling circuit, the lipids being directed to waste).

The now delipidated plasma is drawn by the fluid replacement pump to be mixed with the red blood cells. (A replacement fluid may be added (as required) to the plasma to overcome any loss in bulk of the S plasma during the delipidation and separation steps).

*o 15 As the patient's own blood is used during the method, and no drugs or foreign tissue is introduced, o 0 there should be no rejection of the delipidated blood by the body and there should be no adverse side effects.

The solvent acts to "wash" the lipids from the plasma.

*As previously described with reference to 00 FIG. 2, the lipid levels in the blood drop when the o delipidation method is effected but rise again after o e.g. 3 days apparently due to the stored lipids in the animals body entering the blood stream (see also FIGS.

4 and By repeating the delipidation method at S. intervals, the build up of lipids in the arteries and other organs can be reversed and the likelihood of heart attacks or strokes reduced. As also discussed, the method can be repeated to remove fat-soluble toxins from the body.

The embodjments are described by way of illustrative examples only and various changes and modifications may be made thereto without departing from the present invention defined in the claims.

Claims (11)

1. A method for plasma delipidation of animals using a continuous flow system including: drawing blood from the animal; separating the plasma from the red blood cells; delipidating the plasma with a lipid solvent; remixing the delipidated plasma with the red blood cells; and re-introducing the delipidated blood into the animal.

2. A method as claimed in Claim 1 wherein the delipidation step includes the steps of: mixing the plasma with the lipid solvent; allowing the mixture to separate into an organic solvent/lipid phase and an aqueous delipidated plasma phase; and drawing off the organic phase.

3. A method as claimed in Claim 1 or Claim 2 wherein: the solvent is DIPE preferably peroxide free and preferably mixed with butanol, 1-pentanol and/or 1-hexanol.

4. A method as claimed in Claim 3 wherein: the solvent is butanol-DIPE 40%:60: A method as claimed in any one of Claims 1 to S4 wherein: an anti-coagulant is mixed with the blood before separation; and a replacement fluid solution is added to the delipidated plasma to maintain the blood bulk.

6. A method as claimed in Claim 2 wherein: Sthe organic solvent is separated from the lipids by distillation and is recycled.

7. A method as claimed in Claim 2 or Claim 6 wherein: ether is added to the delipidated plasma to remove any residual solvent or lipids in the plasma, the ether/solvent/lipids mixture being separated from the -1 4c- .1 plasma by centrifugal separation.

8. An apparatus for plasma delipidation of animals including: means to draw blood from an animal; means to separate the plasma from the red blood cells; means to delipidate the plasma using a lipid solvent; means to separate the delipidated plasma from the solvent; means to remix the delipidated plasma with the red blood cells; and means to re-introduce the delipidated blood into the animal.

9. An apparatus as defined in Claim 8 wherein: the means to separate the plasma from the red cells is a first centrifugal separator. An apparatus as defined in Claim 9 wherein: the means to delipidate the separated plasma is a mixer unit containing the lipid solvent Sdrawn from any convenient solvent supply; and (ii) the means to separate the delipidated plasma from the solvent is a primary separator capable o of separating the solvent and lipids into an organic phase and the delipidated plasma into an aqueous phase.

11. An apparatus as defined in Claim 10 further S" comprising a second centrifugal separator which removes any remaining organic phase from the delipidated plasma. 30 12. An apparatus as defined in Claim 8 further comprising means to feed a replacement fluid solution into the delipidated plasma to maintain the blood bulk.

13. An apparatus as defined in Claim 10 further comprising a distillation unit to separate the solvent from the lipids and also comprising a pump means to pump the solvent to the solvent supply for recycling. a 0- M% -I I 1 1 14. A method for the autologous plasma delipidation of animals substantially as hereinbefore described with reference to the accompanying drawings. Apparatus for the plasma delipidation of animals substantially as hereinbefore described with reference to FIG. 6 of the accompanying drawings.

16. A method for the detoxification of fat-soluble toxins from animals substantially as hereinbefore described with reference to the accompanying drawings. DATED this eighteenth day of December 1989. SUNIVERSITY OF QUEENSLAND By its Patent Attorneys GRANT ADAMS COMPANY 4 o I I 4 I 0 1« U e o 6.