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WO2000030669A2 - Utilisation de proteines de transfert des phospholipides plasmatiques pour le traitement de maladies cardio-vasculaires - Google Patents

Utilisation de proteines de transfert des phospholipides plasmatiques pour le traitement de maladies cardio-vasculaires Download PDF

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WO2000030669A2
WO2000030669A2 PCT/NL1999/000720 NL9900720W WO0030669A2 WO 2000030669 A2 WO2000030669 A2 WO 2000030669A2 NL 9900720 W NL9900720 W NL 9900720W WO 0030669 A2 WO0030669 A2 WO 0030669A2
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plasma
hdl
pltp
cholesterol
mice
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WO2000030669A3 (fr
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Marinus Petrus Gerardus De Crom
Teunis Van Gent
Marinus Johannes Van Haperen
Arie Van Tol
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Erasmus Universiteit Rotterdam
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Erasmus Universiteit Rotterdam
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

Definitions

  • the present invention relates to the field of cardiovascular diseases, in particular to the field of diseases associated with elevated plasma levels of cholesterol and/or triglycerides in mammals, particularly in man.
  • Cardiovascular diseases are a leading cause of death, particularly in the western world and especially for men over 45 and women over 65 years of age. World-wide mortality for cardiovascular diseases exceeds 7 million people per annum. Many factors play a role in the aetiology of cardiovascular diseases, such as coronary artery disease. Important ones are hyperlipidaemia, hypercholesterolaemia, hypertension and smoking.
  • Hyperlipidaemia and hypercholesterolaemia are strongly implicated in the development of atherosclerotic plaques, the growth of which leads to impaired blood flow or occlusion of arteries.
  • therapies available were dietary lowering of atherogenic lipids or lipoproteins, possibly applied together with lipid-lowering drugs, such as statins, in high risk patients.
  • lipid-lowering drugs such as statins
  • therapies aim at reducing LDL (low density lipoproteins) cholesterol and triglyceride levels as well as at increasing HDL (high density lipoproteins) cholesterol.
  • Statins are drugs which are capable of lowering LDL cholesterol values by more than 15%, which effect is accompanied by significant reductions in total plasma cholesterol and triglycerides.
  • HDL cholesterol levels are among the best indicators for the risk of atherosclerosis in epidemiological studies (7,8).
  • HDL plasma levels are inversely related to atherosclerosis.
  • HDL are thought to be anti-atherogenic, for example because they mediate efflux of cholesterol from peripheral cells and transport cholesterol to the liver for degradation into bile acids and excretion. This process is known as reverse cholesterol transport (8-10) .
  • Pre ⁇ -HDL a subclass of HDL, are very efficient acceptors of cellular cholesterol in vitro (13-14).
  • Phospholipid transfer protein (PLTP) is a protein which may be involved in pathways towards the generation of pre ⁇ -HDL in plasma (15) .
  • PLTP Phospholipid transfer protein
  • PLTP is thought to be capable of promoting the net transfer of phospholipids between plasma lipoproteins (1-4) and to mediate conversion of HDL (5,6) .
  • Recently a human cDNA for human PLTP has been cloned (16) after which transgenic mice were made encoding human PLTP (17, 18).
  • mice expressed only very low levels of the transgene, and no physiological effects of PLTP could be measured to any reliable extent.
  • the transgene was introduced in a compound transgenic background with apolipoprotein A-I, which makes it difficult, if not impossible to relate any effect to the PLTP transgene.
  • a good and reliable model system is required.
  • the present invention provides such a model system in that transgenic mice are provided which overexpress human PLTP 2.5 to 4.5-fold.
  • transgenic mice are provided which overexpress human PLTP 2.5 to 4.5-fold.
  • the present inventors have found beneficial pharmaceutical effects of huPLTP for the first time and have shown that it is feasible to make pharmaceutical compositions for the treatment of cardiovascular disorders in vivo based on huPLTP.
  • the effects seen in the transgenic mice according to the invention include decreased total plasma cholesterol levels, increased rates of formation of pre ⁇ -HDL and a high plasma capability to prevent cholesterol accumulation and/or improving cholesterol excretion, for example in macrophages .
  • huPLTP is a protein which can be used as a pharmaceutical.
  • a plasma phospholipid transfer protein or a functional derivative or fragment thereof for use as a pharmaceutical. It is of course preferred to apply huPLTP for human applications, in order to avoid an immunological response to heterologous proteins, as it is preferred to apply PLTP of any given species in the treatment of said same species.
  • PLTP herein is defined as any mammalian protein or proteinaceous substance having at least one activity, but preferably all activities of PLTP., which is adopted from Day et al (16). Having the same activity or activities in this respect means that the activities are the same in kind, not necessarily in amount.
  • PLTP or functional fragments or derivatives thereof, can be produced recombinantly, synthetically, in cell free systems, or isolated from a suitable source. It is however preferred to produce it recombinantly, particularly in a eukaryotic host cell, more preferably in a higher eukaryote, such as a mammalian or insect cell. Transgenic animals (such as the mice provided herein) can also be a suitable source for PLTP. Typically, the person skilled in the art is able to choose a suitable expression system for PLTP based on the teachings of e.g. Sambrook, Fritsch and Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989.
  • Functional fragments and/or derivatives of PLTP are defined as such derivatives or fragments which have sequence homology with PLTP over at least a part of the molecule and which share at least the anti-atherogenic properties of PLTP. It is always difficult to say at which level of homology such activity will be present, but the person skilled in the art is capable with present day techniques to identify the regions in PLTP which is necessary for activity, which typically will be the area of the protein in which derivatives need to have a high homology with the original PLTP's for retaining activity. Such a region is of course also the region of interest to derive functional fragments from and to do site-directed mutations in, in order to enhance activities wanted and decrease unwanted activities by recombinant techniques. As a rule of thumb general homology of a derivative and/or a fragment (over the parts that are present) should be recognisable at the amino and/or nucleic acid level by standard homology search programmes and/or by hybridisation.
  • the present invention discloses a first pharmaceutical or medicinal use for PLTP.
  • this first use lies in the field of cardiovascular diseases, especially in the field of preventing or treating atherosclerosis and the results thereof.
  • the invention especially provides uses under conditions of high plasma cholesterol and/or triglyceride levels, were these conditions need to be alleviated.
  • the invention provides the use of a plasma phospholipid transfer protein or a functional derivative or fragment thereof in the preparation of a medicament for the treatment or prevention of medical conditions associated with relatively high plasma levels of cholesterol and/or triglycerides.
  • high cholesterol and/or triglyceride levels are associated with atherosclerosis and its detrimental results, such as stroke and coronary heart disease.
  • the invention provides a pharmaceutical composition for the treatment or prevention of medical conditions associated with relatively high cholesterol levels, comprising plasma phospholipid transfer protein or a functional derivative or fragment thereof and a suitable diluent or other pharmaceutical excipient.
  • PLTP being a proteinaceous substance, it is necessary to prevent breakdown of the active compound in the gastrointestinal tract of a patient to be treated.
  • PLTP proteinaceous substances
  • routes of administration avoiding the stomach such as other enteral or parenteral routes, such as intravenous compositions and the like. Excipients for all routes are by now well known and can be found in any pharmaceutical compendium.
  • Levels of PLTP to be reached in plasma will depend on the condition to be treated and its severity. The upper limit of levels to be reached is of course determined by the patients tolerance or reactions to PLTP. However in general levels in plasma to be reached should be upward from physiological levels, e.g. 1- to 3-fold, or even up to 5- to 10-fold, expressed as units of PLTP.
  • PLTP can be combined in preparations or treatments with conventional or other cardiovascular drugs, such as statins or fibrates.
  • the invention provides a composition according to the invention, which further comprises other agents for the treatment or prevention of medical conditions associated with relatively high plasma levels of cholesterol and/or triglycerides. Typically such agents would be present in these compositions in their usual amounts or lower.
  • the invention further provides a composition for preventing the accumulation and/or improving excretion of cholesterol in a mammal comprising an effective amount of plasma phospholipid transfer protein or a functional derivative or fragment thereof.
  • the invention provides a gene delivery vehicle for delivering a recombinant nucleic acid molecule encoding comprising plasma phospholipid transfer protein or a functional derivative or fragment thereof to a host cell, whereby said recombinant nucleic acid molecule is integrated into the genome of said host cell.
  • Transgenic mice are of course only a means to study possible roles and effects of expressed transgenes. It is of course highly unlikely that any medical treatment for cardiovascular diseases will ever include anything like transgenesis of humans. However, by providing the genome of a host cell, for example one or more liver cells, of the subject with integrated genetic information encoding PLTP activity, the subject obtains a higher capacity of preventing cholesterol accumulation and/or improving cholesterol excretion in the system. This can be done through gene therapy with a gene delivery vehicle.
  • the invention further provides a transgenic host cell obtainable by transfection with a gene delivery vehicle according to the invention.
  • Said transgenic host cell for example a hepatocyte, can be grown and cultivated in vi tro, and when needed, can be used to provide a patient with said cell having increased capacity of preventing cholesterol accumulation and/or improving cholesterol excretion in the system.
  • compositions according to the invention comprise human plasma phospholipid transfer protein, preferably human plasma phospholipid transfer protein having sequence encoded by a cDNA of figure 5. This of course is also true for the uses according to the invention.
  • a human cosmid library was constructed from high molecular weight DNA isolated from blood from a healthy volunteer. This library was screened for cosmids containing the PLTP gene, using human PLTP cDNA (kindly donated by Drs . A-Y. Tu and J.J. Albers) as a probe. Isolated cosmids were mapped using restriction fragments from the cDNA, and a cosmid with approximately 15 kb 5' to the first exon and approximately 3.5 kb 3' to the last exon (19) was selected.
  • Vector sequences were removed by restriction endonuclease digestion and DNA was dissolved in micro-injection buffer (10 mM Tris- HC1, pH 7.5; 0.1 mM EDTA) at a concentration of 1-2 mg/ml .
  • the DNA was micro-injected into fertilized oocytes from FVB mice. These oocytes were transferred into the oviducts of pseudopregnant foster females.
  • Genomic DNA was isolated from tail clips of 10 days old mice, and analyzed for the presence of the HuPLTP transgene by PCR analysis: sense primer: 5 ' -GCCACAGCAGGAGCTGATGC-3 ' ; anti- sense primer: 5 ' -GCGGATGGACACACCCTCAGC-3 ' ; 25-30 cycles (94 ° C, 1 min; 65 °C, 1 min; 72 °C, 1 min) .
  • Transgenic founder mice were bred with FVB mice to obtain transgenic mice.
  • FVB transgenic HuPLTP mice were backcrossed with C57B1/6 mice for four generations. These mice were intercrossed to obtain wild type, hemizygous and homozygous HuPLTP transgenic mice. Animals were kept on regular chow and fasted overnight prior to collection of blood from the orbital plexus.
  • Plasma PLTP activity was assayed using a phospholipid vesicles-HDL system (1, 20) .
  • EDTA-plasma samples 25 ml of 1:75 diluted plasma were incubated with
  • Apo A-I was purified from mouse plasma HDL (density range 1.063-1.21 g/ml) essentially as described (21) and used to immunize rabbits by subcutaneous injection with 100 mg of mouse apo A-I using standard procedures.
  • Total cholesterol was enzymatically determined with the F- Choi kit from Boehringer Mannheim (Mannheim, Germany) after hydrolysis of cholesterylesters with cholesterol esterase from C. cylindraeca (Boehringer Mannheim) .
  • Phospholipids were measured enzymatically using the PAP150 kit from BioMerieux ⁇ (Lyon, France) .
  • Mouse apo A-I was quantitated by a sandwich ELISA, using a polyclonal rabbit anti-mouse apo A-I IgG, performed in 96- well plates coated with this antibody. Purified mouse apo A-I was used as a primary standard. Plasma samples were diluted in PBS-Tween 20 (0.1%)-BSA (0.5%). Bound apo A-I was detected by addition of polyclonal rabbit anti-mouse apo A-I, conjugated to horseradish peroxidase. The assay is linear in the range of 6.5 to 420 ng/ml .
  • the crossed immuno-electrophoresis consisted of agarose electrophoresis in the first dimension for separation of lipoproteins with pre ⁇ - and -mobility. Electrophoresis in the second dimension, i.e. antigen migration from the first gel into an anti-apo A-I-containing gel, was used to quantitatively precipitate apo A-I.
  • Lipoprotein electrophoresis was carried out in 1% (w/v) agarose gels in barbital buffer (50 mM, pH 8.6) and run in an LKB 2117 system (4 °C, 2 h, 250 V) . Five ml of plasma were applied per well.
  • the track of the first agarose gel was excised and annealed with melted agarose to a gel containing 7.5% (v/v) rabbit anti-mouse apo A-I antiserum that was cast on GelBond film (Pharmacia) .
  • the plate was run in an LKB 2117 system (4 °C, 20 h, 50 V) in barbital buffer. Unreacted antibody was removed by extensive washing in PBS.
  • the gel was stained with Coomassie Brilliant Blue R250 and subsequently dried. Areas under the pre ⁇ -HDL and -HDL peaks were calculated by multiplication of peak height and width at half height. The pre ⁇ -HDL area is expressed as a percentage of the sum of a- HDL and pre ⁇ -HDL areas.
  • Pre ⁇ -HDL concentrations are also given in absolute amounts (mg apo A-I present in pre ⁇ -HDL/ml plasma) . These values were calculated from the percentage of apo A-I present in pre ⁇ -HDL and the total plasma apo A-I concentrations .
  • Bovine serum albumin (fraction V grade) was delipidated by extraction of free fatty acids with activated carbon (23) .
  • [9, 10 (n) 3 H]oleic acid (10.0 Ci/mmol, Amersham, U.K.) was complexed to BSA after evaporating 0.1 mmol of oleic acid (48 Ci/mol) to dryness under a stream of nitrogen.
  • AcLDL was prepared from LDL (density range 1.019-1.063 g/ml) , isolated from human plasma by differential centrifugation and subsequently acetylated by repeated additions of acetic anhydride (24). Increased electrophoretic mobility of the acLDL was confirmed by agarose electrophoresis at pH 8.6 (12).
  • C57B1/6 mice were elicited by intraperitoneal injection of 0.8 ml of eliciting agent, prepared from Baker's thioglycollate (Difco) according to manufacturer's instructions. After 4 days, macrophages were obtained as described (25) . The macrophage monolayers were washed with DMEM and incubated with 500 ml aliquots of DMEM that contained 3 mg/ml AcLDL, 0.1 mM BSA- [ 3 H] oleate (48 Ci/mol) and 12.5 times diluted plasma from fasted mice. After 18 h, medium was removed and cells were washed twice with PBS.
  • eliciting agent prepared from Baker's thioglycollate (Difco) according to manufacturer's instructions. After 4 days, macrophages were obtained as described (25) . The macrophage monolayers were washed with DMEM and incubated with 500 ml aliquots of DMEM that contained 3 mg/ml
  • Cholesteryl esters were extracted from the intact monolayers with 1 ml hexane-isopropanol (3:2, v/v) and purified by TLC as previously described (26) . Labeled cholesteryl ester bands were excised from the silica and radioactivity was determined. Protein was extracted from the cell remnants with 0.1 M NaOH and quantified by the method of Lowry et al . (27) using BSA as a standard. Duplicate assays were performed for each plasma sample.
  • trangenic mice A cosmid clone containing the complete human PLTP gene was isolated and analyzed by Southern blotting. It included the 3' end but not the 5' end of the lysosomal protective protein gene. The purified 35 kb cosmid was micro-injected in fertilized oocytes. This resulted in 27 newborn mice, two of which harboured the transgene, as determined by PCR analysis and Southern blotting (data not shown) . Both founder mice were bred into two independent lines of HuPLTP transgenic mice.
  • PLTP activity was measured in plasma samples of mice from two HuPLTP transgenic lines.
  • line #1 PLTP activity in plasma was increased by 281 % compared with the activity in plasma from wild type mice (Table 1), while in line #2 an increase by 262 % was measured (not shown) .
  • Subsequent analyses were performed with line #1 only.
  • the use of either human HDL or mouse HDL as acceptor in the PLTP activity assay showed the same differences between wild type, hemizygous and homozygous transgenic animals, demonstrating that human PLTP interacts both with human HDL and mouse HDL (not shown) .
  • the expression of the transgene was confirmed by Western blotting (Fig. 1).
  • the mice homozygous for the transgene showed a higher plasma protein level of HuPLTP when compared with the hemizygous transgenic mice.
  • no immunoreactive PLTP was detected in wild type mice.
  • Recombinant PLTP was used as a positive control and gave one single immuno-reactive band at a relatively low MW, due to a lower extent of glycosylation in the baculovirus expression system.
  • the transgene was found to be expressed in all tissues analyzed (Fig. 2), with relatively high mRNA levels in adrenal, testis, and lung, and moderate mRNA levels in liver, kidney, intestine, brain and spleen.
  • the tissue pattern of expression of the endogenous PLTP gene was similar to that of the transgene, again with the highest mRNA levels in adrenal, testis, and lung.
  • the expression of endogenous PLTP was not affected by HuPLTP expression in any of the tissues tested (Fig. 2) .
  • the overexpression of PLTP resulted in a decrease in plasma cholesterol levels in the HuPLTP hemizygous transgenic mice and a further decrease in the homozygous transgenic mice
  • Plasma levels of cholesterol, phospholipids and apo A-I were decreased by about the same extent, indicating that the decrease reflects a lowering of HDL, which are the major lipoprotein in mouse plasma. Separation of plasma lipoproteins by gelfiltration confirmed that the decrease in plasma lipids is confined to the HDL fraction (not shown) .
  • Fig. 3 Mouse plasma was analyzed by crossed- immunoelectrophoresis (Fig. 3) .
  • Plasma samples from wild type, hemizygous and homozygous HuPLTP transgenic mice were collected and incubated in the presence of an inhibitor of lecithin: cholesterol acyl transferase (LCAT) in order to prevent maturation of the formed pre ⁇ -HDL into oi-HDL (14).
  • LCAT cholesterol acyl transferase
  • the formation of pre ⁇ -HDL particles is clearly increased in plasma from transgenic mice when compared with wild type mice. It is also evident that the formation of pre ⁇ -HDL is at the expense of -HDL, demonstrating the origin of pre ⁇ -HDL (Fig. 3).
  • Table 2 gives the pre ⁇ -HDL values, both before and after incubation in the presence of LCAT inhibitor. No significant differences are found in freshly frozen plasma between the different genotypes. Before incubation the percentage of pre ⁇ -HDL tends to be highest in the homozygous HuPLTP transgenic animals, but the differences between genotypes are not significant. However, in incubated samples clear differences arise with highest relative and absolute concentrations of pre ⁇ -HDL in the transgenic animals.
  • acylCoA cholesterol acyltransferase
  • ACAT activity determined this way is a measure of the intracellular cholesterol concentration.
  • Mouse peritoneal macrophages were incubated in the presence of [ 3 H] oleate, AcLDL and diluted mouse plasma containing the acceptor HDL particles (see Methods) .
  • Fig. 4 shows that the formation of labeled cholesteryl oleate by ACAT was 25.7+9.7% lower in the presence of hemizygous transgenic plasma as compared to wild type plasma, indicating less accumulation of cellular cholesterol in the presence of plasma from transgenic animals.
  • transgenic mouse plasma has the ability to prevent cholesterol accumulation to a greater extent.
  • ACAT activity compared with wild type plasma, see Fig. 4
  • Plasma phospholipid transfer protein is able to promote the net transfer of phospholipids between plasma lipoproteins (1-4) and mediate conversion of high density lipoproteins (HDL) (5, 6) . Since plasma levels of HDL are among the best indicators for the risk of atherosclerosis in epidemiological studies (7, 8), PLTP could play a role in the prevention of the development of coronary heart disease and stroke via its effect on HDL.
  • HDL are anti-atherogenic because they mediate efflux of cholesterol from peripheral cells and transport cholesterol to the liver, for excretion and degradation to bile acids. This process is known as reverse cholesterol transport (8- 10). It has been postulated that the anti-atherogenic effect of HDL can be attributed mainly to a quantitatively minor subclass of HDL, called pre ⁇ -HDL (11, 12). This assumption is based on in vi tro studies showing that pre ⁇ -HDL is a very efficient acceptor of cellular cholesterol (13, 14). The origin of pre ⁇ -HDL is not well understood, but the available evidence suggests that PLTP participates in its generation, at least in vi tro (15).
  • mice Following the cloning of a human PLTP cDNA (16) , two groups independently generated transgenic mice for human PLTP (17, 18) . Unfortunately, these mice showed low levels of expression of the transgene and, as a result, only small effects on plasma lipoproteins were observed. Changes in HDL levels and subtractions could only be demonstrated in a compound transgenic background with human apolipoprotein (apo) A-I (18) .
  • apo apolipoprotein
  • HuPLTP transgenic mice have been described previously by two other groups, but these models did not show an appreciable overexpression of the transgene.
  • the mice described by Albers et al . (17) showed little expression of the transgene and only small changes in plasma lipoproteins.
  • Jiang et al . (18) reported a 29% increase in PLTP activity, but significant effects on total plasma lipids or lipoproteins were not observed. Only after their HuPLTP transgenic mice were crossbred with mice transgenic for human apoA-I, they detected small effects on plasma lipids and lipoproteins (including an increase in pre ⁇ -HDL levels) , together with a 47% elevation in plasma PLTP activity.
  • HuPLTP has also been overexpressed in mice via adenovirus mediated transfer (30, 31) . These mice showed a 13- to 40- fold elevation of PLTP activity in plasma several days after treatment. This resulted in a dramatic decrease (by 91%) in HDL-levels, while pre ⁇ -HDL levels were substantially elevated (30). These data are in line with our present observations: high PLTP activity results in a decrease in total HDL, while pre ⁇ -HDL levels are increased. It is clear that the effects are transient and greatly exaggerated in the adenovirus treated mice, due to the extremely high plasma levels of PLTP.
  • Plasma of HuPLTP transgenic mice was found to be much more efficient in partially preventing AcLDL-induced accumulation of intracellular cholesterol in cultured macrophages than plasma of wild type mice, in spite of lower levels of total HDL.
  • the most likely explanation for this observation is the increased plasma concentration of pre ⁇ -HDL, which has been identified previously as a very efficient cholesterol acceptor (13, 14).
  • pre ⁇ -HDL which has been identified previously as a very efficient cholesterol acceptor (13, 14).
  • an operative cholesterylester cycle, as present in macrophages (33, 34), is important for cholesterol efflux.
  • the present results imply that the distribution of HDL subclasses is of major importance for the efficacy of HDL-mediated reverse cholesterol transport, even more important than total plasma HDL levels.
  • Plasma levels of total HDL cholesterol are inversely correlated with the incidence of coronary artery disease in man (7, 8) .
  • O'Connor et al . (14) analyzed the steady state levels of pre ⁇ -HDL in 136 normolipidemic individuals, using an isotope dilution technique.
  • Their relative values for pre ⁇ -HDL in human plasma are quite comparable with the values measured in mouse plasma (see Table 2).
  • the percentage pre ⁇ - HDL (percentage of total plasma apo A-I) was negatively correlated with total HDL cholesterol concentrations, in line with our observations in mice. Plasma PLTP activity was not measured in their study.
  • Reconstituted HDL particles enriched in triglycerides which are model particles for HDL prevalent during alimentary lipemia, are more rapidly converted by PLTP to pre ⁇ -HDL than triglyceride-poor HDL (35) .
  • This observation suggests that hypertriglyceridemia may be associated with increased generation of pre ⁇ -HDL by PLTP.
  • Syvanne et al . (36) reported a positive correlation between PLTP activity and the capability of plasma from patients with diabetes mellitus and coronary heart disease to induce cholesterol efflux from Fu5AH rat hepatoma cells.
  • mice over-expressing human PLTP.
  • the lines huPLTPl and huPLTP4 have been generated using a cosmid containing the entire gene plus its natural flanking sequences (fig. 6) and thus express PLTP driven by its native promoter (mice) . These mice have different levels of plasma activity of PLTP (see table 3) .
  • mice as well as wild type mice were fed a high sucrose diet for 2 weeks (LFC; 40) and subsequently a high fat, high cholesterol diet (HFC/0.5%; 40 ; containing 1% cholesterol and 0.5% cholate; Hope Farms, Woerden, The Netherlands), which is a standard diet to study diet-induced atherosclerosis in mice (41) .
  • HFC/0.5%; 40 containing 1% cholesterol and 0.5% cholate; Hope Farms, Woerden, The Netherlands
  • Analysis after 4 weeks on this diet of the plasma lipoproteins by gel chromatography (figure 7) showed that cholesterol was lowered in all lipoprotein fractions when compared with plasma from wild type mice, including the atherogenic VLDL/IDL fraction. The lowest cholesterol levels were found in the PLTP4 mice, that have the highest PLTP activity.
  • PLTP protects against diet-induced increase of atherogenic VLDL/IDL.
  • Atherosclerotic lesions in the mice were analyzed after 16 weeks of HPC/0.5% diet. The area of aortic atherosclerotic lesions was smaller in mice with higher levels of plasma PLTP activity.
  • PLTP protects against diet-induced increase in plasma cholesterol levels, and protects against the development of diet-induced atherosclerosis.
  • mice over-expressing either human PLTP (huPLTP mice) , human cholesteryl ester transfer protein
  • CETP huCETP mice
  • huPLTP/huCETP mice Both the activity of PLTP as well as formation of pre ⁇ -HDL in plasma were studied. It appeared that the activity of plasma PLTP was equal in wild type compared to huCETP mice. Also, in huPLTP compared to huPLTP/huCETP mice, plasma PLTP activity was equal. The formation of pre ⁇ -HDL in plasma (fig.
  • PLTP determines the rate of formation of plasma pre ⁇ -HDL, independent of CETP levels.
  • HuAITg mice are less susceptible to diet-induced atherosclerosis in comparison with wild type mice (42) .
  • Plasma PLTP activity is higher in the huAITg mice than in wild type mice (Fig. 9) .
  • Fig. 10 Plasma from huAITg mice was more effective in this respect.
  • Fig. 1 Western blot analysis of the plasma levels of HuPLTP in transgenic mice. SDS-PAGE was carried out in 12.5 % (w/v) gels, proteins were electrophoretically transferred to PVDF membranes and visualized with a rabbit polyclonal antibody raised against a synthetic peptide with identity to amino • acids 470-493 of HuPLTP. Goat anti-rabbit IgG conjugated to peroxidase was used as a secondary antibody. Antigen-antibody complexes were visualized by chemiluminiscence using the ECL system (Amersham) . Blots were exposed for 20 sec to a Kodak XAR-5 film. Re refers to recombinant PLTP from a baculovirus expression system. Plasma from wild type mice and hemizygous and homozygous HuPLTP mice is indicated by wt, he and ho, respectively.
  • RNA levels of either the transgene (HuPLTP) or the endogenous, murine gene (MuPLTP) were examined by gel electrophoresis of RT-PCR products. RT-PCR on HPRT was used as a loading control.
  • FIG. 3 Apolipoprotein A-I immunoprecipitation patterns of plasma from wild-type or HuPLTP transgenic mice obtained after crossed immunoelectrophoresis .
  • the figure shows a representative display of the pre ⁇ - and -HDL bands obtained by analysis of mouse plasma, incubated for 3 h at 37 °C in the presence of iodoacetic acid (1 mM) , an inhibitor of LCAT. Samples were then analyzed by crossed-immunoelectrophoresis as described in Methods. In order to confirm mono-specificity of the anti apo A-I antibody towards ⁇ -HDL and pre ⁇ -HDL fractions, we performed immunoelectrophoresis as described (28).
  • Fig. 4 Esterification of cholesterol by ACAT in peritoneal macrophages incubated simultaneously with AcLDL, [ 3 H] oleate and plasma of wild type (Wt) , hemizygous (He) or homozygous HuPLTP transgenic mice (Ho) .
  • Plasma was obtained from age- matched wild type (Wt), hemizygous (He) and homozygous (Ho) HuPLTP transgenic mice.
  • Peritoneal macrophages were incubated for 18 h in the presence of 3 mg/ml AcLDL, 0.1 mM [ 3 H]oleate- BSA (48 Ci/mol) and diluted mouse plasma.
  • Esterification by ACAT is expressed as dpm in cholesteryl oleate/mg cell protein.
  • a decreased esterification indicates a decreased cellular cholesterol concentration.
  • Fig. 5a Nucleotide sequence and derived amino acid sequence of human PLTP. Data as reported by Day et al. (1994) (GenBank accession number L26232). The 30 C-terminal amino acids have been reported to be indispensable for phospholipid transfer activity (Huuskonen et al . , 1998).
  • Fig. 5b PLTP cDNA sequence and alignment with several proteins. The upper line contains the nucleotide sequence of human PLTP cDNA as reported by Day et al. (1994) (GenBank accession nubmer L26232) . PLTP belongs to a family of proteins that are structurally and functionally related (Hubacek et al., 1997).
  • CETP Human cholesteryl ester transfer protein (GenBank accession number M30185)
  • LBP Human lipopolysaccharide binding protein
  • Fig. 6 Schematic drawing of the PLTP and lyosomal protective protein (LLP) genes, that are on opposite DNA strands. The sequences included in the cosmid used to generate transgenic mice, are indicated
  • Fig. 8 Pre ⁇ -HDL formation in plasma from wild-type (W) , huCETP mice (C) , huPLTP mice (P) or huPLTP/huCETP mice (CP) .
  • W wild-type
  • C huCETP mice
  • P huPLTP mice
  • CP huPLTP/huCETP mice
  • the figure shows a representative display of the pre ⁇ - and ⁇ - HDL bands obtained by analysis of mouse plasma, incubated for 3 h at 37°C in the presence of iodoacetic acid (1 mmol/L) , an inhibitor of LCAT. The positions of pre ⁇ - and ⁇ -HDL migration are indicated.
  • Values are means ⁇ SD. Values between brackets are percentage of the wild type values. For further details see Methods.
  • Plasma PLTP activities were measured in pooled plasma from 5-6 mice and expressed as percentage reference plasma

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Abstract

L'invention concerne le domaine des maladies cardio-vasculaires, en particulier celui des maladies associées à des concentrations plasmatiques élevées du cholestérol et/ou des triglycérides chez les mammifères, en particulier chez l'homme. Selon l'invention, la protéine de transfert des phospholipides plasmatiques (PLTP) présente des effets pharmaceutiques bénéfiques et il est possible de produire, sur la base de la PLTP, des compositions pharmaceutiques pour le traitement d'affections cardio-vasculaires.
PCT/NL1999/000720 1998-11-24 1999-11-23 Utilisation de proteines de transfert des phospholipides plasmatiques pour le traitement de maladies cardio-vasculaires Ceased WO2000030669A2 (fr)

Priority Applications (1)

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AU14168/00A AU1416800A (en) 1998-11-24 1999-11-23 Use of plasma phospholipid transfer proteins in treating cardiovascular dieases

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EP98203962 1998-11-24
EP98203962.0 1998-11-24

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WO2000030669A2 true WO2000030669A2 (fr) 2000-06-02
WO2000030669A3 WO2000030669A3 (fr) 2000-10-19

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US5622843A (en) * 1993-12-30 1997-04-22 Zymogenetics, Inc. Phospholipid transfer proteins and DNA encoding them
US5932536A (en) * 1994-06-14 1999-08-03 The Rockefeller University Compositions for neutralization of lipopolysaccharides

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