CA2146481A1 - Method for immunization of mammals against atherosclerosis and pharmaceutical compositions for obtaining said immunization - Google Patents

Abstract

A method for immunizing mammals, particularly humans, against atherosclerosis, and pharmaceutical preparations for the prophylaxis and treatment of atherosclerosis. The pharmaceutical preparations are obtained by subjecting certain lipoproteins, derived from either the blood of the patient or from the same mammalian species, to an in vitro fusion step to provide heavy microemulsion particles. The fused proteins are then incorporated into a pharmaceutically acceptable immunization carrier or diluent for delivery to the patient.
These compositions avoid the complexities of presently available methods of treatment, and appear to be more effective. The lipoproteins used are those known as VLDL, LDL, and VLDL-remnant or IDL.

Description

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A METHOD FOR IMMUNIZATION OF MAMMALS AGAINST ATHEROSCLEROSIS
AND PHARMACEUTICAL COMPOSITIONS FOR OBTAINING SAID IMMUNIZATION
Field of the Tnvention The present invention relates to a method for immunizing mammals, particularly humans, against atherosclerosis. The present invention also relates to pharmaceutical compositions for the treatment, for prophylaxis of atherosclerosis, and to a method for the preparation of said pharmaceutical compositions.
S ~ ry o~ the Invention The present invention relates to a method for the immunization of a mammal against atherosclerosis comprising the following steps (a) obtaining a sample of mammalian blood, from a mammal of the same species as the mammal to be treated;
(b) isolating a lipoprotein from the blood sample;
(c) transferring the isolated lipoprotein to a suitable buffer system;
(d) causing the lipoprotein in the buffer system to fuse, to provide heavy microemulsion lipoprotein particles;
(e) recovering the fused lipoprotein heavy emylsion particle3;
(f) if desired incorporating the heavy microemulsion particles into a pharmaceutically acceptable immunization carrier or diluent; and (g) administering to the mammal the microemulsion particles obtained in step ( f ) .
In a first broad embodiment this invention seeks to provide a ph:~rTn~-eutical composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosls, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and a 21~g~1 pharmaceutically acceptable immunization carrier or diluent therefor.
Preferably, in the pharmaceutical composition the fused lipid materials are heavy microemulsion particles obtained by the fusion step. More preferably, the fused lipoprotein material is chosen from at least one member of the group consisting of fused very low density lipoproteln (VLDL), fused low density lipoprotein (LDL), fused int~ ;Ate density lipoprotein (IDL), and mixtures thereof.
In a second broad embodiment the present invention seeks to provide a method for the preparation of a pharmaceutical composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosis, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and, if desired a pharmaceutically acceptable ; 7Ation carrier or diluent therefor, which process comprises inducing fusion of the ~1 ;An lipoprotein in vitro in a suitable buffer system either by adding an amphipathic fusogenic material to the - l; An lipoprotein, or by exposing the mammalian lipoprotein to osmotic stress, if reriuired removing the fusogenic material, recovering the fused mammalian lipoprotein, and, if desired, incorporating the recovered fused lipoprotein in a pharmaceutically acceptable; ;7Ation diluent or carrier.
In a third broad embodiment the present invention seeks to provide a method for the preparation of a ph~rr~ceut; cal composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosis, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and, i~ desired a phArr~reutically acceptable ; zation carrier cr diluent therefor, which process comprises:
(a) obtaining a sample of mammalian blood, from a mammal of the same species as the mammal to be treated;

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(b) isolating a lipoprotein from the blood sample;
(c) transferring the isolated lipoprotein to a suitable buffer system;
(d) causing the lipoprotein in the buffer system to fuse, to provide heavy microemulsion lipoprotein particles;
(e) recovering the fused lipoprotein heavy emulsion particles, and if desired incorporating the heavy microemulsion particles into a pharmaceutically acceptable i7At;on carrier or diluent.
Preferably the lipoprotein is caused to fuse either by exposing it to the effects of a fusogenic material, or by exposing it to osmotic stress. ~ore preferably, a fusogenic material comprises an effective amount of a fusogenically active peptide.
The invention further relates to a method for the treatment of atherosclerosis in mammals according to a conventional i 1 7ation scheme, comprising admlnistering an initial dosage of the above-mentioned pharmaceutical compositions to the rn l; ~n body, and subsequently administering further dosages of the same composition to the mammalian body over a desired time period, such as every two weeks.
Backqround of the Invention Atherosclerosis is a degenerative disease affecting the entire human population. It is brought about by a change in the normal structure of the blood vessels, especially in the areas known as lesion-prone, located at vessel branching points, or in the regions where the blood flow is turbulent due to the morphology of the human circulatory system. This change is represented by an accumulation of extracellular lipids, A('l_ ~ Ation of lipid-laden macrophages (foam cells), proliferation of vascular smooth muscle cells and an accumulation of extracellular matrix. All these changes reduce the vascular 21~6~81 .
lumen and may finally obstruct the vascular flow, with the known pathological consequences.
A portion of the population that is especlally at rlsk are the familial hypercholesterolemics, who have a defective apolipoprotein B receptor. In this genetic disease, there is an increased quantity of circulating low density lipoproteins (LDL), the main cholesterol carrier in the human body. In addition, the lipoproteins with a pre-~ electrophoretic migration pattern, such as very low density lipoproteins ~VLDL) and the floating ~-lipoproteins (,~-VLDL) also have a high atherogenic potential (Bates, S.R., J.Lipid.Res., 1987,28, 787) . A further important atherogenic lipoprotein is intermediate density lipoprotein (VLDL
remnant, or IDL) (Shaikh, M. et al, Arterioscl.Thromb., 1991, 11, 569). Clearance of cholesterol from the circulation is impaired due to the defective receptor. Consequently, there is also an increased transport of these proteins such as typically LDL into the vessel intima, by non-receptor mediated transport. The excess of lipoprotein in the vessel intima of these patients gives rise to increased frequency of atherogenic lesion formation and an increase in the dimensions of these lesions.
The treatments now in use for this disease are intended (i) to lower the circulating cholesterol level (and, implicitly the LDL or related protein circulating level); this is the case with lipid-poor diets and with lipid-lowering drugs and (ii) to counteract the modifications which are considered to make the LDL
or related proteins more atherogenic; this i5 the case with antioxidants of the probucol and carotene type. The more extreme case of the genetic hyperlipidemias may require a treatment by the two above procedures, but combined with reducing the circulating cholesterol level by LDL apheresis (Scholzek, P. et al. Clinical Investigator, 1992, 70, 99) .
The present proposed method for atherosclerosis treatment is based on an entirely different principle, implying an immunization against the first form of lipid reorganized

2~ gl m~t~rj~l appearing in the lesion-prone areas AS a consequence, the immune system reacts very rapidly against the lipid material in the lesions, which is taken out by a monocyte clearance system based on an immune mechanism. Moreover, the existence of an immune-based ---ch~niqm for lipid clearance causes a reversion of the lesion-prone areas to their normal state, irrespective of the circulating cholesterol level. This is achieved in a time which is approximately equivalent to 1/lOOth of the lesion development time, and the balanced situation of the vessel is m~;nt~;n~rl from this point on.
Detailed DescriPtion of the Invention The reorganized lipid material in the atherogenic lesions according to the present invention is formed by a lipid fusion process of a protein from the group including VLDL, LDL
and IDL in the vascular intima. Taking LDL as a typical example, the fusion process produces heavy microemulsion droplets with increased dimensions and multiple apolipoprotein B copies on their surface, a situation in which new antigenic det~rm;n~ntq of the apolipoprotein B are exposed, rendering the heavy microemulsion droplets antigenic and immunogenic.
The fusion of the lipoproteins of the group including VLDL, LDL, and IDL, was obtained in vitro in simple conditions not implying the modification of the native state of the lipid material as regards its oxidation state, and reproducing simple ionic conditions most pro~ably existing in an intima undergoing an atherogenic transformation. By immunizing experimental normal animals such as hamsters with this material containing only the reorganlzed lipid material derived from LDL in physiological saline, it was posslble to select specific B and T lymphocyte clones capable of reacting agalnst a similar type of reorganized LDL materlal, immediately after its formation in the leslon-prone areas of the anlmals on an atherogenic diet. The Syrian golden hamster was used (Nistor, A., et al., Atherosclerosls, 1987, 68, 159), because of the great similaritles between its lipoprotein 21~6~
metabolism and the human lipid metabolism ~Spady, D.K., et al., Proc. Natl. Acad. Sci., USA, 1983, 80, 3499). Moreover, this animal was shown to develop obstructive coronary lesions at long diet times. Moreover, lipoproteins with the characteristics of VLDL have been isolated from atherosclerotic human aortas (Rapp, J.H., et al, Aterioscl. Thromb. 1994, 14,1767) .
The important advantage of this procedure is that the reorganized form of the lipid protein, such as VLDL, LDL, and IDL, exists only in the lesional areas, and these areas are the only target of the immune-competent lymphocyte clones selected by the ~ i 7~tion. By the lmmunization a reversion of the affected lesion-prone areas to the normal structure is induced and maintained. This management of the atherogenic leslons can be applied both prophylactLcally and therapeutically, as will be detailed below. The normal metabolism for the unreorganised form of the lipoproteins is not affected, since the circulating form of the lipoprotein is the unmodified form.
The lipoprotein fusion in vlvo is a lipoprotein modification leading to an increased capacity of the native lipoprotein based material to generate atherogenic lesions. The essence of the present invention is the development of a lipoprotein fusion procedure in vitro, leading to a reorganised form of the material which is identical to the in vlvo material.
This type of material is both antigenic and immunogenic (i.e., it represents a non-self structure which has the capacity to select specific lymphocyte clones producing antibodies against it). This statement is based on the reaction of the immune system against the extracellular lipid material in the lesions, when the immunization and the diet were applied sequentially.
The VLDL, LDL, and IDL lipoproteins are easily isolated and characterised. The following procedure for LDL fusion is exemplary .

21~81 Blood was obtained from the experimental animals by cardiac puncture. From human subjects (blood donors) the blood was obtained by phlebotomy under standard conditions. Plasma was immediately obtained from the blood samples by low speed centrifugation. The LDL fraction was obtained from fresh plasma by single vertical spin density gradient ultracentrifugation (Chung, B.H., et al., J. Lipid Res., 1980, ~L 284) . The LDL
purity was checked by immunochemical methods using affinity-purified anti-holoLDL polyclonal antibodies. The native state of the LDL was checked by the high performance liquid chromatography (HPLC) quantitation of its molondialdehude (MDA) content. The level found characterized the isolated lipoproteln as native (Berliner, J.A., J. Clin. Invest., 1980 85, 1260). The integrity of the LDL apolipoprotein B, characterizing its native state, was also checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The in vitro fusion step can be carried out by several procedures, as several substances have fusogenic capabilities.
The peptides bacitracin, and vasopressin share common structural characteristics including a hydrophobic ring structure, and a positively charged amino group c~ntZ~;n~ng end. These important characteristics are also present in the neuropeptide somatostatin, and in the vasoconstrictor endothelin peptides.
Somatostatin is a tetradecapeptide, with a ring ~ ncl ll~i; n~ 12 amino acid groups, and 2 amino acids forming a carboxy-~rm;n;~l end. The endothelins comprise 21 amino acids, with a cyclic structure and an amphipathic carboxy end (see Janes, R.W. et al, Nat. Struct. Biol., 1994, 1, 311). Endothelin is known to be a marker for arterial disease.
The fusion step is typically carried out as follows.
The lipoprotein is transferred into half-diluted phosphate buffered saline (PBS) containing 72 mM Na+,2 mM K~, 0.3 nM Mg2', and from 0.45 mM Ca2l, by geL filtration on a 2 ml S~rhA~ G-25 (trade mark) column. The concentration of Ca2' is raised to 1. 6 ~ 2146~1 mM. These ionic rrnr~ntrations are characteristic for the physlological saline solution used as a fusion buffer (FB). This buffer was found to be essential for the fusion procedure. The lipoprotein is then incubated at 20C with a suitable amount of the fusogenic reagent, typically for a time period of from 15 to 20 minutes. Thereafter the fused lipoprotein is recovered by any suitable means, and, if desired, combined with a ph~rr~re~ltically acceptable; ;7atlon diluent or carrier for administration purposes .
It has also been found that the in vitro fusion process can be applied to mixtures of lipoproteins: for example a mixture of human VLDL and LDL has been successfully treated as a heterogeneous system.
It has also been found that although it is preferred to use blood from the patient to be treated by immunization with the fused lipoprotein to provide the required material, this is not necessary. Blood from another source of the same species of mammal can also be used in the fusion process to provide the material for immunization.
One of the unique properties of fusogenic materials, such as bacitracin, is a pronounced amphipathic activity. By the term "amphipathetic" is meant a molecule with different characteristic properties, particularly a combination of hydrophillic and hydrophobic properties. By utilising these properties, the fusogenic material can be removed from the fused preparation by a simple dialysis, due to its small molecular weight. After the dialysis the total removal of the fusogenic material from the preparation can be ascertained by any suitable analysis method, such as a suitable quantitative E~PLC method.
It has also been found that the fusion process can be induced, at least with LDL, without using a fusogenic material.
It has been found that fusion can be caused by an osmotic stress 2~ 4~81 .
inducing the fusion process by osmoelastic coupling. The osmotic stress was generated by subjecting the LDL, transferred in FB, to a dialysis against 209r polymer solutions of polyethylene glycol ~PE;G) 20, 000 or dextran 40, 000 .
B~ief descriDtion o~ the Drawinqs.
In the following experimental data reference ls made to the attached drawings in which:
Figure 1 shows graphically the fusion of hamster derived LDL;
Figure 2 shows graphically the fusion of human derived VLDL, mixed VLDL and LDL, and LDL;
Figure 3 shows graphically comparative fusion curves for human derived LDL with different fusogenic materials;
Figure 4 shows the effects of negative staining on LDL
particles;
Figure 5 shows the stabilisation of lipid material in immunised animals;
Figure 6 shows the reversion of lesions in the immunised animalsi Figures 7, 8 and 9 show further tissue changes in the immunised animals, and Figures 10 and 11 show potassium bromide ultracentrifugation results for the materials of figures 2 and

3.
ExDerir~ 1 Data The in vi~ ro work below was done for both animal and human material. Only the immunization work was done solely in animals .
Fugion ~;~tho-lc.
Example 1. Fusion of hamster LDL by bacitracin.

2146~81 .
To 0.1-0 7 mg/ml LDL in FB, bacitracin was added from a fresh 70 mM solution to a final concentration of 0.35 mM, and the preparation was lncubated at 200C for 15 min. The best fusion yield was obtained at a bacitracin to apolipoprotein B
ratio of 7.5 to 1. At the end of the incubation period the Ca2' was chelated with ethylene diamine tetraacetic acid (EDTA) (2mM
final concentration). An extensive dialysis of the preparation against 4,000 volumes of PBS followed. This dialyzedpreparation was used for the immunization.
Example 2 Fusion of hamster LDL by vasopressin.
To 0.1-0.7 mg/ml LDL in FB, vasopressin ~Sigma Chemical Co., USA) was added from a fresh 1 mg/ml solution at the final concentration of 2 pg/ml and the preparation was i n~ h~ted at 200C for 30 min. An extensive dlalysis against 4, 000 volumes of PBS followed.
Example 3. Fusion of hamster LDL by osmotic stress.
An 0 .1-0 . 7 mg/ml LDL solution in FB was subjected to a 15 min. dialysis at 20C against a 20% solution of PEG 20, 000 or dextran 40, 000 in water. The cutoff limit of the dialysis bag was 10,000. An extensive dialysis against 4,000 volumes of PBS
followed .
Example 4. Fusion of human VLDL by bacltracin.
Heavy emulsion particles were obtained at 20C from purified human VLD~ in FB using a concentration of 0 . 88 mM
bacitracin. The incubation period was 17 min. The formation of the heavy microemulsion particles was monitored by gel filtration, turbidimetry, and ultracentrifugation in a KBr gradient. The turbidimetry results are shown in Figure 2, and the KBr ultracentrifugation results in Figure 10. Gel filtration analysis was performed using a Sephadex g-150 column, which had 2~46481 been calibrated for the elution volumes with purified human lipoproteins including VLDL and LDL. The turbidimetry data indicates that a plateau is reached after about 17 min. The ultracentrifugation analysis showed that a 1.24 g/ml band was obtained after VLDL fusion, which indicates the formation of heavy micro emulsion particles.
Example 5. Fusion of mixed human VLDL and LDL by bacitracin.
Using the same conditions as in Example 4, above, a mixture of isolated purified VLDL and 1DL was treated, at a concentration for each lipoprotein of 0.1 mgtml. The fusion again took about 17 min (see Figure 2), and on ultracentrifugation a band at 1.24 mg/ml was obtained.
Example 6. Fusion of human LDL with somatostatin and endothelin-1.
To demonstrate the fusogenic action of both somatostatin and endothelin-1 the procedure of Example 4 was employed, and the same types of assays made (see Figure 3).
Purified human LDL at 0.15 mg/ml was treated in the same FB
buffer, and was incubated at 20C with bacitracin, vasopressin, somatostatin, and endothelin-1, with a final concentration of the fusogenic agent of 60 M. Gel filtration again indicated an increase in particle dimensions in each case. Curves of turbidity as a function of time each show a plateau reached after about 15 min. incubation. The ultracentrifugation results differ a little (see Figure 11) . For bacitracin, there are two bands, at 1.24 and 1.20 mg/ml, whilst for the other three materials there are two bands with flotation densities between 1.17 and 1.20 mg/ml. These results collectively indicate that somatostatin has a fusogenic capability comparable to that of vasopressin, whilst endothelin-l is more similar to bacitracin.
In the fusion buffer employed, both somatostatin and endothelin-1 ~t4~81 transform human LDL particles into heavy microemulsion particles of increased dimensions.
Bacitracin produced the LDL fusion in the presence of 1. 6 mM Ca2~ in 7 min.; the equilibrium was attained for hamster LDL in 15 min. and for human LDL in 20 min. This was indicated by the turbidity of the sample, monitored spectrophotometrically by absorption at 400 nm as is shown in Figure 1. Bacitracin was effective in inducing the LDL fusion from 0.35 mM to 0.35 pM.
The concentration of vasopressin tested was in the physiological range, at 2 pg/ml.
The fusion phen~ -n~ were observed ultrastructurally by negative staining. The LDL preparation was formed from 20 nm particles of uniform size (see Figure 2A), floating at 1.06 g/ml in the gradient density ultracentrifugation (see Figure 2B) . The fused preparation was formed from microemulsion droplets with dimensions ranging from 30 to 300 nm (see Figure 2C), floating in two bands at 1 21 and 1.24 g/ml (see Figure 2D). The dimensions, and the flotation densities, of the microemulsion droplets in the fused preparations were similar to the material isolated from the experimental hamster atherogenic lesions. It was also concluded that the change in the flotation density in both in vivo and in vitro material was due to the presence of multiple copies of apolipoprotein B on the surface of the microemulsion droplets. This conclusion was based on a correlative investigation of both in vivo and in vitro material from the experimental animals and of in vitro studies of the human material; this correlative investigation comprised biochemistry, cytochemistry, immunochemistry, electron microscopy and work in vitro with isolated cells (hamster monocytes) .
The heavy microemulsion particles proved to have the same pattern of organization as the LDL: a core of neutral lipid stabilized in a hydrophillic medium by a phospholipid monolayer (see Figure 2E ) in which multiple copies of the apolipoprotein 2146~81 .
B were embedded. By taking the LDL parameters as a ~asis, an estimation of the droplet surface/LDL surface ratio would predict 2 copies of apolipoprotein B on the surface of a 30 nm particle and 225 copies on the surface of a 300 nm particle. This novel rearrangement of self molecules proved to be antigenic and immunogenic .
Animals were immunized by a conventional immunization scheme comprising a primary immunization with 50 g of protein/animal (mean weight 100 g) and three secondary i 7ations at two-week intervals with 25 ug of protein each, with the micro^ 1 c; r~n obtained in vitro; a group was hyperimmunized and received 7 secondary immunizations. Then, these animals were given a hypercholesterolemic diet in the form of standard hamster chow supplemented with 5% cholesterol and 1596 butter, for 7, 14, 30 and 60 days. After these diet times, 2-3 ml of blood were obtained by cardiac puncture for biochemical and immunochemical investigation, then the animals were sacrificed for the ultrastructural investigation of their lesions.
The total cholesterol determinations in the sera of these animals indicated clearly their hypercholesterolemic state, by comparison with the values obtained from the atherogenic control animals. The only significant deviation from the control values was at 7 and 14 days of diet, when a significant increase in the level of circulating cholesterol was observed. The e~planation of this increase in the level of circulating cholesterol was due to an immunosuppression phenomenon because of the unfused LDL present in the preparation. This immunosuppression was not present in the case of animals immunized with fused human LDL (Table 1).

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T~hle 1.
Cholesterol values in the experimental atherogenic animals (mg/dl) (pooled sera) ormal Control atheroqenic Immunized Hyperimmunized Immunized with human fused Ll~L
103 ~t7 150 (7 days diet) 327 ~72 126 ~38 200 (14 days diet) 419 i:73 146 i:16 250 (30 days diet) 258 ~t49 240 *76 244 *13 300 (60 days diet) 352 *96 After this initial increase the cholesterol level dropped and reached values comparable to that of the atherogenic ~n i ~ zed control .
The circulating antimicroemulsion antibody titers determined by conventional enzyme-linked immunosorbent assay (E1ISA) showed measurable, but very low, antibody titers ~2 to -3) only at 7 days of diet. At all the other diet times, no measurable levels of circulating antibodies were detected, indicating that the immune reaction was localized to the lesional areas, as will be detailed below. The cross reactivity of the sera from the antigen challenged animals (immunized animals receiving an atherogenic diet) with the homologous LDL was tested and a negative result was obtained.
The effect of the immunization was tested on lesion development in the region of the aortic valve rings. This area 214 64~1 .
was particularly susceptible to the atherogenic diet, developing fibrolipidic like lesions 10 times faster than any other lesion-prone areas in the hamster. The main body of these lesions was formed by the extracellular lipid material in a very dynamic state of lipid component rearrangement. In the antigen-challenged animals at 7 days of diet, the lesions developed in the aortic valve ring area, implying that the LDL metabolism in these animals was not affected, being similar to tne control atherogenic animals. At this time of diet, in the controls, the extracellular lipid material was in the form of densely packed microemulsion droplets in a dynamic state of reorganization. In the antigen challenged animals the microemulsion lipid material was stabilized (see Figure 3A) and numerous plasma cells surrounded the lipid deposits (see Figure 38). They had characteristic distensions of their endoplasmic reticulum, indicating an active antibody synthesis (Figure 38, inset). The reaction of the immune system to the microemulsion droplet material in the lesions in the antigen challenged animals implied an identity between the microemulsion droplet material formed in vivo in the lesions and the in vitro material used for the immunizations, since the diet was administered post-immunization.
At 14 days of diet, the former fibrolipidic-like lesions developed in the lln; ~ zed controls were replaced by a fatty streak (see Figure 4), implying that a reversion of the lesion type took place. This was not previously induced in any eXpPr~r^nt;~l model of atherogenesis by any type of experimental manipulation and is a process only presumed to take place naturally in humans in which, occasionally, lesional areas requilibrate (Munro, J.M. Cotran, R.S Lab. Invet., 1988, 58, 249) . At 30 and 60 days of diet, 80% of the aortic ring area had an aspect close to the normal. Moreover, the clearance of the area was assured by numerous monocyte-derived from cells, showing morphological sp~r~li7ations which clearly indicated a close cooperation in their egression ~rom the tissue (see Figures 5A
and 5B).

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In the animals immunized with the fused human 1DL the same trend of events was observed, and the presence of antibody secreting B cells was the same around the lesions. Consequently the same reversion events took place, and at about 1 month of diet the vessel returned to its normal structure.
In the hyperimmunized animals, at 30 days of antigen challenge post-; i 7~tion, the structure of the aortic ring area was normal, as compared to the normal animals, which were not i ; zed and received a normal diet, with the immunized controls which were immunized animals receiving a normal diet ~see Figures 6A, 6B, 6C) .
In a special group of animals, the immunization was applied post-diet to elucidate if the same reversion process could be induced. After 3 months of atherogenic diet these animals had a total plasma cholesterol of 281 ~:95 mg/dl. By taking into consideration the moment of activation of the resident valvular macrophages into presenting the antigen, a process which appears from the first week of diet in the antigen-challenged animals and appears very late ~6 months) in the course of atherogenic lesion development (Filip, D.A., et al.
Atherosclerosis, 1987, 67, 199) a working hypothesis indicated that a reasonable moment to check the eventual reversion was at 1/10 of the total diet time from the moment the ~ l 7~tion was completed (the immune surveillance system established). A
considerable degree of reversal was obtained in comparison with the atherogenic controls (see Figure 7A) . However, the previous diet period left sequelae, representing the effects of lipid component reorganization (see Figure 7B) . Nevertheless, even the reorganized lipid material in the form of micronic cholesteryl ester droplets (see Figure 7B) was taken up by the monocyte/macrophages in the same time with the phagocytosis of the heavy microemulsion formed ~n vf vo by the fusion . The mechanism of disposal of cholesteryl ester micronic droplets was similar to the one observed in v~t~o during the incubation of 2~4~81 monocyte/macrophages with cholesterol crystals (Koren, E. et al., Prgo. Lipid Res., 1991, 30, 237) . It consisted in the surrounding of the cholesteryl ester micronic droplets with multiple phospholipid by layers (see Figure 7B).
In conclusion, it appears that the immunization against a heavy microemulsion formed by the LDL fusion and representing the initial step in the extracellular lipid accumulation in the atherogenic lesion-prone areas, reversed the atherogenic evolution by blocking its initial step. This immunological approach proved beneficial for the management of atherogenic lesions even if applied in advance or post-diet. Therefore, this approach may represent even a prophylactlc or a therapeutic method of atherogenesis treatment by an immunological ---h;lr1.cm which is a vaccination.
Quantitative studies regarding lipoprotein transfer in human intima have shown that another important atherogenic lipoprotein is an intP ~ te density lipoprotein, known as VLDL
remnant, or IDL. Since both VLDL and LDL can be fused either by means of a fusogenic material or by osmotic stress, and IDL
particles are an intermediate form between VLD~ and 1DL no reason is seen why IDL should not behave similarly. IDL is known to have dimensions and a radius of curvature close to that of LDL.
The fusogenic capabilities of bacitracin, vasopressin, somatostatin and endothelin-1 all point to a structural similarity of these substances, to a class of peptide compounds capable of causing lipoprotein fusion. For example, both endothelin-2 and endothelin-3 are expected to have the same fusogenic properties as endothelin-1, as their structures are closely similar. The structuraL properties of these molecules suggest that other peptides are also fusogenic materials.
Detailed Description of th~ Fic~ure~

21~481 Figure 1. The process of hamster LDL ~usion by bacitracin, monitored as absorptLon at 400 nm (black circles).
Each value represents the mean of triplicate aliqotes. As shown by the kinetics, the fusion is complete in 7 minutes and the equilibrium is attained in 9 minutes. For the human LDL the kinetics is somewhat slower, the fusion being complete in 15 min.
and the equilibrium attained in 20 min. The same type of measurements were done for the initial and final absorption values in osmotic stress-induced fusion experlments (trianqle), also for hamster LDL.
Figure 2. The process of human VLDL, equal amounts of mixed VLDL and LDL, and LDL fusion is shown monitored in the same way as in Figure 1. In each case, the lipoprotein total concentration was 0.2 mg/ml, and the fusing peptide was bacitracin, at a final concentration of 0 . 88 mM. As the curves show, a plateau is reached in 9 to 11 minutes.
Figure 3. The process of human LDL fusion by four fusogenic materials is shown monitored in the same way as in Figure 1. The four fusogenic materials are bacitracin, somatostatin, vasopressin, and endothelin-1. As the curves show, a plateau is reached in 9 to 11 minutes. The curves also show that bacitracin and endothelin-1 show strong fusogenic activity, whilst vasopressin and somatostatin are less active.
Figure 4A. The LDL particles aspect in negative staining: the particles have similar dimensions around 20 nm (X
95, 400) -Figure 4B. The LDL, separated by a single verticalspin density gradient centrifugation has a flotation density around 1. 0 6 g/ml .

2t46~81 Figure 4C. ~he negative staining aspect of the LDL
fused preparation, in which microemulsion droplets have dimensions between 30 and 100 nm (X 181, 400) .
Figure 4D. The microemulsion droplets, due to their high protein content (multiple apolipoprotein B copies on their surface) float at much higher densities (1.20 and 1.24 g/ml).
Figure 4E:. The microemulsion droplets have the same pattern of organization as the lipoprotein, being composed of a core of neutral lipids (evidenced here by a cytochemical reaction with tannic acid-paraphenylene diamine), stabilized by a phospholipid monolayer (X 95, 400) .
Figure 5A. In the immunized animals, starting with the first week of hypercholesterolemic diet post-; l7at;on, the lipid material in the microemulsion form is stabilized in comparison with the control hypercholesterolemic animals, in which the same type of material i~ in a dynamic state of lipid component reorganization (X 95,400).
Figure 5B. Numerous plasma cells (x 29, 400), by ultrastructural criteria, surround the extracellular accumulations of lipid material; these cells have the characteristic distended rough endoplasmic reticulum, indicative of antibody synthesis (inset) (X 59, 600) .
Figure 6. At 14 days of diet, the previous fibrolipidic like lesions are transformed into fatty streaks.
No more extracellular lipid material in microemulsion form is found extracelluarly. This demonstrates the reversion of the previous fibrolipidic-like lesion to a more benign form of atherogenic lesions, namely the fatty streak.
Figure 7A. At 30 days of diet, the foam cells are massively egressing from the tissue (X 8, 300) .

21~81 .
Figure 7B. The foam cells have specialized interdigiti2ed structure, assuring a cooperative action in the region of the endothelial cell junctions (X 29, 400) .
Figure 8A. In the case of hyperimmunized animals, at 30 days of diet post-immunization, the lesional area is already re-equilibrated between the microemulsion droplet material formation and its disposal by the foam cell egression, resulting in a normal aspect of the lesion-prone area (X 54, 600) .
Figure 8B. The aspect of the same area as in Figure 6A
in the normal control animals, in which some microemulsion droplets can be seen immediately under the endothelial cells;
their presence reflects the very high propensity of this area to develop atherogenic lesions (X 37, 800) .
Figure 8C. The same region in the control immunized animals, not receiving an atherogenic diet (x 54, 600) .
Figure 9A. The aspect of the lesions in the animals receiving the immunization post-diet. The ; 1 7ation was started after 3 months of atherogenic diet, and the animals were sacrificed at a total diet time of 17 weeks (at an l/lO total diet time after the; 17at;on scheme was completed). The lesion regression is evident, though the sequelae of the initial period of diet are present, especially in the form of cholesteryl ester droplets (X 54, 600) . However, even this type of material was disposed off by the monocyte/macrophages in the region, by a characteristic type of phagocytosis.
Figure 9A. (inset) The immediately above portion of Figure 9A enlarged, at X 18, 600 .
Figure 9B. The aspect of the lesions in the atherogenic controls, at approximately the same diet time (x 3, 600) .

6~81 Figure 10. Potasgium bromide gradient ultracentrifugation results are shown, for LDL, mixed LDL and VLDL, and VLDL matching the urbidity data in Figure 2. A 1.24 mg/ml band is obtained in all three cases, for which the fusogenic material was bacitracin, at O . 88 mM final concentration .
Figure 11. Similar potassium bromide gradient ultracentrifugation results to those in Figure 10 are shown for the comparison of bacitracin, vasopressin, somatostatin, and endothelin-l .
The fusogenic capability of the peptide is shown to be directly related to the increasing densities of the heavy microemulsion particles obtained.

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosis, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and a pharmaceutically acceptable immunization carrier or diluent therefor.

2. A pharmaceutical composition according to claim 1 wherein the fused lipid materials are heavy microemulsion particles obtained by the fusion step.

3. A pharmaceutical composition according to Claim 1 wherein the fused lipoprotein material is chosen from at least one member of the group consisting of fused very low density lipoprotein (VLDL), fused low density lipoprotein (LDL), fused intermediate density lipoprotein (IDL), and mixtures thereof.

4. A method for the preparation of a pharmaceutical composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosis, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and, if desired a pharmaceutically acceptable immunization carrier or diluent therefor, which process comprises inducing fusion of the mammalian lipoprotein in vitro in a suitable buffer system either by adding an amphipathic fusogenic material to the mammalian lipoprotein, or by exposing the mammalian lipoprotein to osmotic stress, if required removing the fusogenic material, recovering the fused mammalian lipoprotein, and, if desired, incorporating the recovered fused lipoprotein in a pharmaceutically acceptable immunization diluent or carrier.

5. A method for the preparation of a pharmaceutical composition for treatment of atherosclerosis, and for prophylaxis of atherosclerosis, including as active ingredient a fused lipoprotein material from the same species as the mammal to be treated and, if desired a pharmaceutically acceptable immunization carrier or diluent therefor, which process comprises:
(a) obtaining a sample of mammalian blood, from a mammal of the same species as the mammal to be treated;
(b) isolating a lipoprotein from the blood sample;
(c) transferring the isolated lipoprotein to a suitable buffer system;
(d) causing the lipoprotein in the buffer system to fuse, to provide heavy microemulsion lipoprotein particles;
(e) recovering the fused lipoprotein heavy emulsion particles, and if desired incorporating the heavy microemulsion particles into a pharmaceutically acceptable immunization carrier or diluent.

6. A method according to Claim 5 wherein in step (a) the mammalian blood is human blood.

7. A method according to Claim 5 wherein in step (b) the isolated lipoprotein is chosen from at least one member of the group consisting of fused very low density lipoproteins (VLDL), fused low density lipoproteins (LDL), fused intermediate density lipoproteins (IDL), and mixtures thereof.

8. A method according to Claim 5 wherein in step (c) the buffer comprises half-diluted phosphate buffered saline containing 72 mM Na+, 2 mM K+, 0.3 nM Mg2+, and 1.6 mM Ca2+.

9. A method according to Claim 5 wherein in step (d) the lipoprotein is caused to fuse by exposure to osmotic stress.

10. A method according to Claim 9 wherein the osmotic stress is obtained by subjecting the buffer containing the lipoprotein to dialysis against a solution of a water soluble polymer.

11. A method according to Claim 10 wherein the water soluble polymer is chosen from polyethylene glycol having a molecular weight of about 20,00 or dextran having a molecular weight of about 40,000.

12. A method according to Claim 5 wherein in step (d) the lipoprotein is caused to fuse by exposure to an effective amount of a fusogenic reagent.

13. A method according to Claim 12 wherein the fusogenic reagent is a peptide.

14. A method according to Claim 13 wherein the peptide is chosen from the group consisting of bacitracin, vasopressin, somatostatin, and an endothelin.

15. A method according to Claim 14 wherein the endothelin is endothelin-1.

16. A method according to Claim 5 wherein the fused lipoprotein is recovered by a dialysis step.