Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to an endogenous baboon plasma cholesteryl esters transfer protein (CETP) inhibitor poiypeptide. More specifically, this invention relates to the identification and characterization of the poiypeptide and to novel synthetic peptides possessing inhibitory activity of CETP.
• the endogenous inhibitory peptide has a molecular weight of 4000, is present in plasma in the form of modified apo A-l and apo E having molecular weights of 31kD and 41kD, respectively, and has a common amino acid sequence with the N-terminal fragment of apo C-1.
• This invention also relates to an anti-atherosclerosis composition, a kit, and to antibodies raised against the N-terminal amino acid sequence of the inhibitory poiypeptide.
• the inhibitory peptide of the invention, fragments thereof and analogues thereof are useful for the prophylactic and therapeutic treatment of atherosclerosis.
• Atherosclerosis is one of the most widespread health problems in the United States today as are its attendant complications, particularly coronary heart disease.
• a number of risk factors have been associated with the development of premature atherosclerosis, primarily elevated plasma cholesterol levels.
• Due to the crucial role cholesterol appears to play in the occurrence of heart disease a great deal of attention has been devoted to studying its synthesis, transport and metabolism in the human body.
• Of particular interest is the establishment of relationships between the levels of plasma lipoproteins or serum lipids and the risk of development of coronary heart disease.
• Both high density lipoproteins (HDL) and low density lipoproteins (LDL) carry cholesterol mainly in the form of cholesteryl esters (CE).
• HDL cholesterol is an even more important negative risk factor. Although the exact functions of these lipoproteins have not been completely established, HDL appears to serve for the removal of cholesterol from peripheral cells and its transport back to the liver, where a large proportion of the cholesterol excreted from the body is removed.
• LDL and HDL are believed to play key roles in the development of cardiovascular disease by overloading the lysosomes of the walls of arterial cells with metabolites which are generally hydrolyzed slowly, such as CE and triglycerides. These products are evacuated from the liver and intestine by plasma LDL. When the amount of lipids to be transported exceeds the transporting capacity of HDL to the liver for excretion, CE become deposited in the cells in certain critical areas, such as arterial walls. This overloading eventually results in impaired cell function, and if continued may produce cell death. A continuous overloading results in the accumulation of cellular debris and the formation of atherosclerotic plaque in the vessel wall.
• lipoproteins of a density intermediate to that of LDL and HDL, or large high density lipoproteins. These lipoproteins have been designated HDL j and the animal phenotype as "high HDLi”.
• Baboon strains possessing, for instance, patterns of either high or low HDL X are known. In most cases, HDL X separates either as a distinct peak between LDL and HDL or as a shoulder to the HDL peak, and is induced by a high cholesterol, high lard (HCHF) diet.
• HCHF high cholesterol, high lard
• the proportion of HDL j diminishes when the baboons are fed a diet that is either enriched in polyunsaturated fat, with or without cholesterol. Occasionally, however, the amount of HDL j present in high HDL X baboons fed the chow diet is low. In some baboon families, the level of plasma HDL X was shown to increase when the animals are challenged with a HCHF diet. When fed a HCHF diet, the baboons also show higher plasma HDL. More generally, the accumulation of HDL in baboons as well as in humans is associated with a slower transfer of CE from HDL as very low density lipoproteins (VLDL) and LDL. Thus, baboons with high HDL ! plasma levels are excellent as animal models for the study of hyperalphalipoproteinemia.
• VLDL very low density lipoproteins
• HDL is generally divided into subfractions based on their particle sizes and densities. These fractions include HDL j , HDL 2 , and HDL 3 .
• HDL X has the largest particles and is usually not present in the plasma of normal humans or non-human primates.
• HDL 2 and HDL 3 are the normal components of human plasma.
• HDL 2 is larger than HDL 3 and differs between men and women.
• U.S. Patent No. 4,987,151 to Taboc discloses triterpene derivatives that inhibit acyl coenzyme Axholesteral acyltransferase (ACAT) enzyme.
• ACAT is a cellular enzyme that is not present in plasma, and esterfies cellular cholesterol to form CE. This enzyme is different from the CE transfer protein (CETP) present in plasma.
• CETP CE transfer protein
• the CETP does not form CE as does the ACAT enzyme. Instead, the CETP transfers CE amongst different plasma lipoproteins.
• U.S. Patent No. 4,643,988 to Segrest, et al discloses amphipathic peptides which are capable of substituting for apo A-I in HDL.
• Apo A-I is known to stimulate the lecithin cholestero acyl transferase (LCAT) enzyme, a plasma enzyme that forms CE in HDL.
• LCAT lecithin cholestero acyl transferase
• Plasma CETP in contradistinction, transfers CE from HDL to VLDL and LDL.
• the function of the CETP enzyme is, therefore, different from that of the LCAT enzyme, as well.
• the amino acid sequence of the Segrest et al peptides are, in addition, different from the sequences of the CETP inhibitor of this invention.
• This invention relates to a substantially pure poiypeptide having activity inhibitory of CE transfer protein (CETP).
• CETP CE transfer protein
• This invention also relates to an anti-atherosclerosis composition, comprising an anti- atherosclerosis effective amount of the poiypeptide described above; and a pharmaceutically-acceptable carrier.
• this invention relates to an anti-atherosclerosis kit, comprising in separate containers at least one unit of the composition described above; at least one syringe; and at least one needle.
• This invention also relates to an antibody having specificity for a poiypeptide selected from the group consisting of the poiypeptide described above; baboon CETP poiypeptide inhibitor; 1-36 amino acid N-terminal fragment of apo C-I; modified apo A-I (NW:31kD); modified apo E (MW:41D).
• this invention relates to a method of preventing atherosclerosis in a mammal being predisposed to that condition, comprising administering to the mammal a prophylactically effective amount of the poiypeptide described above.
• This invention also relates to a method of treating a mammal afflicted with atherosclerosis comprising administering to the mammal a therapeutically effective amount of the poiypeptide described above.
• HDL 2+3 collects cholesterol from extrahepatic cells, which is then esterified by LCAT to form cholesteryl esters (CE) and stored in the core of the particles.
• the HDL becomes larger in size (HDL j ) and may pick up apo E to attain a particle which is removed from LDL receptors (LDL-R) on liver cells.
• the CE enriched HDL X may also donate CE to VLDL and LDL. This is mediated by CETP. Due to the presence of the CETP inhibitor, such as the one provided herein, CE transfer is slow (bar) and the reciprocal transfer of triglycerides (TG) does not take place (X).
• the triglyceride-poor HDL is thus not a suitable substrate for hepatic triglyceride lipase (HTGL). Due to the presence of the CETP inhibitor, in plasma, VLDL and LDL are thus not available to the liver. As a consequence of this, the liver then increases the expression of messages for the increased production of LDL receptor and 3 hydroxy, methyl, glutaryl-coenzyme A (HMG-COA) synthase.
• HMG-COA glutaryl-coenzyme A
• LDL receptor in the liver leads to an increase in uptake of LDL or HDL, with apo E, and consequently, to a greater delivery of cholesterol to the liver.
• An increase in synthesis of HMG- CoA synthase leads to an increase in synthesis of cholesterol in the liver to meet all cellular needs.
• the presence of a CETP inhibitor in plasma will prevent the uptake of VLDL and/or LDL by tissues as well as the deposition of cholesteryl esters.
• HDL high levels of HDL have an anti-atherosderogenic effect whereas high levels of LDL have an atherogenic effect.
• the circulation in blood of compounds, such as cholesterol, that are insoluble in water requires the formation of particles.
• the insoluble components e.g., cholesteryl esters and triglycerides, are packed in the core of the partides and surrounded by polar components such as proteins, phospholipids, and the like. These particles are called lipoproteins, and have, thus, an outer polar shell and a non-polar core.
• VLDL is the largest lipoprotein secreted by the Hver and is converted into IDL, and then to LDL, after the triglycerides contained by the VLDL are hydrolzyed by the lipoprotein lipase enzyme present on the surface of the arterial walls.
• LDL is the major lipoprotein that provides cholesteryl ester to extrapepatic and hepatic tissues.
• HDL is also secreted by the hver and is divided in HDL 2 and HDL 3 on the basis of size and density.
• a function of HDL is to pick up cholesterol from extrahepatic cells and deliver it to the Hver, either through VLDL and LDL or through large HDL which is enriched with apo E.
• These large HDL partides are called HDL : and they do not stay in the plasma for long periods of time. They are rapidly removed by the Hver or converted back to HDL 2 after donating their cholesteryl esters to VLDL and LDL.
• HDL X is not present in either normal humans or non-human primates. As indicated above, HDL ! appears as a distinct band in the plasma of baboons that have been fed a HCHF diet. From what is known, when cholesterol enters the blood stream it becomes assodated with HDL in the form of a CE, with the help of the LCAT enzyme. In HCHF-fed baboons, this appears as an HDL r CE fraction. The CE is then transferred from HDL to VLDL and LDL to form VLDL-CE and LDL-CE with the aid of the CETP enzyme. These partides then enter the Hver ceUs through the LDL receptor (LDL-R).
• LDL-R LDL receptor
• VLDL-CE After being metabolized in the Hver ceUs, the VLDL-CE is returned to the plasma and thus to the periphery of the mammalian body, where its deposition may occur leading to atherosclerosis.
• An inhibitor of CETP such as the one provided by this invention, blocks the transfer of CE from HDL to VLDL and LDL. Instead, a shunt is favored that leads to the association of the CE with apo E and to the formation of HDL j -CE-apo E particles that can enter the Hver ceUs through the LDL receptor (LDL-R).
• the apoHpoprotein C-I of various spedes but not baboon, are known.
• the Apo C-I is a single poiypeptide of molecular weight 6600, consisting of 57 amino adds. It is a basic protein that is mainly present in VLDL and HDL, with HDL serving as a reservoir for this protein.
• LDL contains Httle apo C-I. It has recently been shown that apo C-I displaces apo E from VLDL and affects its binding to the LDL receptor.
• the poiypeptide inhibitor of CETP that is described herein has a common sequence with the N-terminal fragment of apo C-I. This fragment indudes at least 36 amino acids as shown below.
• the endogenous poiypeptide (SE . ID. NO: 1) provided by this invention has a molecular weight of about 4,000 and becomes assodated or binds to apo A-I and apo E in plasma. Its N-terminal 36 amino adds are shown below.
• a poiypeptide having the sequence corresponding to amino adds 1 to 36 of the foUowing sequence (SEQ. ID. NO: 1) was synthesized by the inventors and shown to be inhibitory of CETP in vitro.
• the peptide has the foUowing sequence.
• Fragments of that poiypeptide comprising the C-terminal fragments of amino acids 28 to 36 and amino adds 16 to 36 showed Hmited inhibitory activity of CETP at 50 ⁇ g.
• the fragment comprising the C-terminal amino adds 28 to 36 showed at 200 ⁇ g an activity inhibitory of CETP approximately the same as that of the 36 amino add peptide.
• the N-terminal fragments comprising amino acids 1 to 15, amino adds 1 to 20 and amino adds 1 to 10, as weU as the intermediate fragments comprising amino adds 15 to 30 and the like, corresponding to the synthesized peptide have been shown active as inhibitors of CETP.
• Polypeptides having the sequences corresponding to the foUowing two sequences designated (SEQ. ID. NO:2) and (SEQ. ID. NO:3) have also been synthesized by the inventors and shown to be inhibitors of CETP in vitro.
• the first of the foUowing two sequences (SEQ. ID. NO:2) is a baboon sequence like (SEQ. ID. NO:l) except that it has two additional amino adds at the beginning of the peptide.
• the second of the foUowing two sequences (SEQ. ID. NO:3) is a human sequence and varies from (SEQ. ID. NO:2) in seven of the thirty-eight amino adds in the sequence.
• Analogues of the poiypeptide of the invention and fragments thereof having inhibitory activity of CETP are also part of the invention.
• the analogues may have one or more substitutions in their sequences while stiU preserving their inhibitory activity.
• Examples of analogues suitable as inhibitors of CETP are analogues of the peptide of the invention and fragments thereof, such as those where one or more of the amino acids are substituted in accordance with the guidelines provided below.
• the substitute amino acids may be selected from the group consisting of
• the poiypeptide is capable of inhibiting the binding of an about 31kD modified apo A-I poiypeptide present in the plasma of high HDL j baboons or a peptide of the sequence.
• the poiypeptide of the invention is capable of inhibiting the binding of an about 4kD CETP inhibitor poiypeptide present in the plasma of high HDL j baboons to an antibody raised against a peptide of the formula
• the poiypeptide of this invention is capable of inhibiting the binding of an about 41kD modified apo E poiypeptide present in the plasma of high HDL j baboons with antibody raised against the 36 amino acid N-terminal fragment of apo C-I or a peptide of the formula
• the poiypeptide is capable of inhibiting the binding of the 36 amino add N-te ⁇ ninal fragment of apo C-I or a peptide of the formula
• Preferred polypeptides are the polypeptides of the sequence 1 2 3 4 5 6 7 8 9 10 11 12
• Gly ⁇ -Ala, C ⁇ -methylAla, and 2-amino butyric acid for Ala; norLeu, isoLeu, and C ⁇ -methylLeu for Leu; ornithine, Arg, dtrulHne and C ⁇ -methylLys for Lys; Ala and 2-amino isobutyric acid for Gly;
• the poiypeptide contains amino adds 1 through 36 of the above sequence (SEQ. ID. NO. 1).
• the peptide is selected from the group consisting of peptide fragments (SEQ. ID. NO. 1) comprising amino acids 1 to 17, 1 to 20 and 1 to 25, and fragments thereof having anti-4kD peptide antibody/1-36 amino add peptide binding inhibitory activity.
• the peptide fragments are selected from the group consisting of peptides comprising amino adds 1 to 18, and 1 to 28 of (SEQ. ID. NO. 1), and fragments thereof having anti-4kD peptide antibody/1-36 amino adds peptide binding inhibitory activity.
• analogues with the Lys, Asp and Asn amino adds substituting for the Arg, Glu, and Gin amino adds; the Ser, Leu, and Ala amino acids substituting from the Thr, lie and Gly amino acids; the ornithine, dtrulHne and a aminoadipic add amino adds substituting for the Lys and Glu amino adds.
• analogues SEQ. ID. NO. 1.
• Peptides comprising amino add sequences where amide bond(s) (e.g., -C(O)-NH-) linking any pair, and up to aU pairs, of amino acids comprising amino acids 1 to 17, 1 to 20, 1 to 25, 1 to 36, and fragments thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inhibitory activity are replaced by thioether bonds (e.g., -CH 2 -S-) alkyl such as ethyl (-CH 2 -CH 2 -), and/or amino (e.g., - CH 2 -NH 2 -) linkages.
• thioether bonds e.g., -CH 2 -S- alkyl such as ethyl (-CH 2 -CH 2 -)
• amino e.g., - CH 2 -NH 2 -
• analogues are also part of this invention as long as they preserve the inhibitory activity of the antibody/1-36 amino add peptide binding.
• the CETP inhibitory poiypeptide of the invention may be provided as a powder, preferably in freeze-dried form, as a solution, preferably frozen at below -20°C, and the like, to prevent proteolysis.
• This invention also provides an anti-atherosderosis composition, comprising an anti-atherosclerosis effective amount of the poiypeptide of the invention; and a pharmaceuticaUy-acceptable carrier.
• the composition When the composition is used as preventative tool, it may contain 10 to 200 mg and more preferably 20 to 100 mg of the poiypeptide. However, other amounts are also suitable.
• the amount of poiypeptide present is preferably about 10 to 400 mg, and more preferably about 20 to 300 mg. However, other amounts may also be utilized.
• composition of this invention may be provided in unit form, preferably in a sterile, closed container, and more preferably in a sealed container.
• a kit comprising in separate containers at least one unit of the anti-atherosderosis composition of the invention; at least one syringe; and at least one needle.
• kits may contain from about 1 to 20 units of the composition of the invention, but could contain 50 units or more.
• the kit may contain 1 to 20, but and sometimes 50 or more syringes if they are disposable, and 1 to 20, but sometimes up to 50 or more needles if they are disposable.
• the components of the kit are provided in a sterile form, be it wrapped in a sealed, sterilized wrapping, or in some other way. If not disposable, the syringe and needle may be autodaved between uses.
• the composition of the invention is preferably administered intravenously, although it may also be administered intraperitoneaUy, subcutaneously or intramuscularly.
• the oral route is not permissible since the poiypeptide would be degraded in the addic pH of the stomach.
• the composition may preferably have a pH of about 7 to 9, and more preferably about 8 to 9, which may be adjusted with the addition of a base, add or buffer as is known in the art.
• This invention also provides an antibody having specificity for a poiypeptide selected from the group consisting of the polypeptides of the invention; the baboon CETP poiypeptide inhibitor, fragments thereof and analogues thereof; the 1-36 amino add N-terminal fragment of apo C-1; modified apo A-I (MW:31kD); and modified apo E (MW:41kD).
• the antibodies of the invention may be raised in mammals as is known in the art. (Albers, J.J. Hazzard, W.R., Immunochemical Quantification of the Human Lp(a) Lipoprotein, Lipids 9:15-26 (1974)).
• the antibodies may be raised in rabbit, goat, sheep, pig, and chicken. However, other mammals may also be utilized. Preferred are rabbit antibodies. Also preferred are polychonal antibodies. However, monoclonal antibodies may also be prepared by methods known in the art (Kohler, G., and MUstein, D., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity Nature (London) 256:495-497 (1975)). In one preferred embodiment, the antibody of the invention is capable of specifically binding to modified apo A-I.
• the antibody is capable of specificaUy binding to the baboon CETP inhibitor polypeptides of this invention. In another preferred embodiment, the antibody of the invention is capable of specificaUy binding to the poiypeptide of the sequence.
• the antibody is capable of specificaUy binding to modified apo E.
• the antibody of the invention in another preferred embodiment is also capable of specificaUy binding to apo C-1, and more preferably to the 1-36 N-terminal fragment thereof.
• a method for preventing atherosderosis in a mammal being predisposed to that condition.
• the method comprises administering to the mammal a prophylacticaUy effective amount of the poiypeptide of the invention, or of the anti-atherosderosis composition described above.
• the poiypeptide is administered in an amount of about 2 to 100 mg for preventative appHcations. However, other amounts may also be administered.
• the poiypeptide or composition thereof may be administered in a small volume of carrier, e.g., 0.2 to 1.5 ml of saline or other carriers, as is known in the art.
• the poiypeptide of the inventions may be administered intravenously, to a fragment of the population, particularly the human population, that is not afflicted by high blood cholesterol and hyperbetaHpoproteinemia, but, as detennined by other means, may be at risk of being afflicted by atherosclerosis.
• One such example may be a familial trait having been determined.
• the poiypeptide of this invention may be administered on a daUy basis, or at longer intervals if provided as a slow release composition as is known in the art, such as depoestradiol provided by the UpJohn Co. (UpJohn Co., Kalamazoo, MI).
• the present invention provides a method of treating a mammal afflicted with atherosclerosis. The method comprises administering to the mammal a therapeuticaUy affective amount of the poiypeptide of the invention.
• the poiypeptide When administered for therapeutic purposes, the poiypeptide may be injected in an amount of about 10 to 400 mg, and more preferably 20 to 300 mg. However, other amounts as assessed by a practitioner in specific cases, may also be administered.
• the polypepti.de may be administered intravenously, among other routes.
• the monkey chow is low in fat (10% of total calories) and high in carbohydrate (62% of total calories). In addition, the chow has a very low cholesterol content (0.03 mg Kcal).
• the high HDL j baboons were progeny of two sires (X1672 and X102) who had a high HDL j phenotype.
• the low HDL j baboons were progeny of a number of sires who did not have a high HDL j phenotype.
• the presence of HDL X was detected by high performance Hquid chromatography (HPLC) as described previously (WilHams, et al., Detection of Abnormal Lipoprotein in a Large Colony of Pedigreed Baboons Using High-Performance Gel Exclusion Chromatography, J. Chromatography 308:101-109 (1984)).
• High and low HDL j baboons were immobilized with 10 mg/kg of ketamine HC j and bled.
• the blood was coUected in tubes containing 1 mg/ml EDTA, and plasma separated by low speed centrifugation at 6°C.
• the plasma was treated with sodium azide, choloramphenicol, gentamycin sulfate, phenylmethyl-sulfonyl fluoride and DTNB as described previously (Kushwaha, R.S., et al., Impaired Plasma CE Transfer with Accumulation of Larger High Density Lipoproteins in Some Families of Baboons (papio sp.), J. Lipid Res. 31:965-973 (1990)).
• the total and free cholesterol contents of HDL 3 were measured prior to use in the assay.
• VLDL+LDL from low HDL j baboons was used as the acceptor of CE from HDL 3 .
• a VLDL+LDL fraction of d ⁇ 1.040 g/ml was isolated from 100-200 ml of blood by sequential ultracentrifugation as described previously (Kushwaha, et al. (1986), supra).
• Total and free cholesterol contents in acceptor Hpoprotein were measured by enzymatic methods (Wako Pure Chemical Co.) (AUain, CC, et al., Enzymatic Determination of Total Serum Cholesterol Clin. Chem. 20:470-475 (1974)).
• AU Hpoprotein fractions and the Hpoprotein-deficient fraction of d>1.21 g/ml (LPDS) were dialyzed against saline/EDTA.
• the LPDS was used as the source of CETP.
• Fgflm le A Cholesteryl Ester Transfer Assay
• CE transfer activity of a sample was assayed by a modification of a procedure described previously (Kushwaha, et al., (1986), surpa).
• 3 H CE-labeled HDL 3 containing 50-100 ⁇ g of CE from low HDL ! baboons was incubated with VLDL+LDL containing 100- 300 ⁇ g CE in the presence of LPDS.
• the acceptor Hpoprotein and the LPDS were obtained from low HDL j baboon plasma (chow diet).
• the HDL 3 was obtained from high HDL j baboons maintained on the HCHF diet.
• the incubations were carried out at 4°C (control) and 37°C for 4-6 hrs. and terminated by placing the samples on ice.
• the assay mixture was then ultracentrifuged to separate VLDL+LDL having a d>1.040 g/ml, and the radioactivity in the Hpoprotein was counted as described previously (Kushwaha, et al. (1986), supra). Any difference observed in the radioactivity transferred from HDL to VLDL+LDL at 4°C and 37°C was attributed to CETP activity in the LPDS. Time course experiments gave a linear response up to 7 hrs.
• the CETP activity was linear with increasing LDPS, up to 140 ⁇ l LPDS, which was derived from an equivalent volume of plasma. They poiypeptide of the invention, and synthetic fragments thereof were added to the reaction mixture to determine their CETP inhibitory activities, along with an appropriate synthetic control peptide. The percent difference between the control experiment and the assay with inhibitor peptide was expressed as the inhibitor activity.
• the bottom non-Hpoprotein fraction obtained by ultracentrifugation for 72 hrs was analyzed for protein content by 10% SDS-polyacrylamide gel electrophoresis (LaemmH, U.K., Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227:680-685 (1970)), without ⁇ - mercaptoethanol pretreatment.
• Hpoproteins dilapidated Hpoproteins of d ⁇ 1.21 g/ml from high and low HDL j baboons were separated by 15-19% SDS-polyacrylamide gel electrophoresis with -mercaptoethanol pretreatment prior to loading the samples onto the gels.
• a dialysis tube of molecular weight cut-off point 1000 was attached to the bottom of the gel tube to receive the electroeluted peptide.
• the thus electroeluted peptide was dialyzed and quantitated by comparing its absorbancy at 660 n with a known amount of stained albumin electroeluted at the same time.
• Example 7 Antibody Preparation
• the apoHpoproteins were separated by 15% SDS-gel electrophoresis and stained (LaemmH, (1970), supra). The stained bands were transferred onto a nitroceUulose membrane. The bands corresponding to the inhibitor poiypeptide were cut out (0.05 mg) and dissolved in 0.5 ml of filtered DMSO. 0.5 ml Freund's adjuvant were then added and thoroughly mixed and the mixture was injected intradermaUy into two rabbits. After 30 days, the rabbits were boosted with a simUar amount of electroeluted protein band. Antibody titer was measured by Western blotting. The rabbits were boosted again 3 times.
• Sepharose beads (Pharmacia Co.).
• the bound Hgand was IgG predpitated from the serum of rabbits having antibodies.
• the method used to predpitate IgG was similar to that described by McKinney and Parkinson (McKinney, M.M. and Parkinson, A., A Simple, Non-Chromatographic Procedure to Purify Immunoglobins From Serum and Ascites Fluid, J.Immunological Methods 96:271-278 (1987)).
• the IgG was dialyzed overnight in 100 volumes of phosphate buffered saline, and then dissolved in sodium acetate buffer, pH 8.3, coupled to 3 g of CnBr-activated Sepharose beads, and maintained in Tris-saline, pH 7.4, until ready to use.
• the bound proteins were eluted with 0.1 M acetic acid, pH 3.0 and 1 ml aHquots were coUected and read at 280 nm to visualize the peak.
• the protein fraction was dialyzed immediately against phosphate buffered saline and separated by electrophoresis in 15%
• Example 9 Immunoblotting The proteins separated with SDS-polyacrylamide gels were transferred onto ImmobUon-P sheets (Millipore, Beford, MA). The sheets were incubated with antibody against inhibitor peptides after blocking of nonspecific sites. The sheets were washed and incubated again with a secondary antibody containing horseradish peroxides. The addition of boric acid buffer containing 3-amino-9- ethylcarbozole, methanol and hydrogen peroxide produced a coloration.
• the peptides were synthesized by soHd-phase peptide synthesis as described by Barany and Merrifield (Barany, G. and Merrifield).
• the 1-36 amino add synthetic peptide was assembled from the C-terminus towards the N-terminus, with the ⁇ -carboxyl group of the amino add attached to a sohd support and was then characterized by HPLC
• the CETP inhibitory activity was lost from the HDL when the Hpoproteins were extensively ultracentrifuged, e.g., for 72 hrs, or by repeated ultracentrifugation. After ultracentrifugation, the inhibitory activity was found in the infranatant fraction.
• the infranatant fraction (d ⁇ 1.21 g/ml) was separated by 10% SDS polyacrylamide gel electrophoresis in the absence of -mercaptoethanol.
• the infranatant fraction from high HDLj baboons contained albumin, a protein sHghtly larger than apo A-I and another protein larger than apo E.
• Lanes A and B show nonHpoprotein fractions from high and low HDL X baboons, respectively.
• the protein bands, 1, 3, and 5 correspond to albumin, apo E and apo A-I, respectively.
• Protein samples from high HDL X baboons show only bands corresponding to albumin, a protein of molecular weight 41,000 and a protein of molecular weight 31,000. The molecular weights were determined with standard proteins separated on similar gels (gel picture not shown).
• the infranatant fraction from low HDL j baboons contains these proteins as weU, but in addition, it also contains apo A-I and apo E. Both proteins in the apo A-I region were identified by immunoblotting with antibody to apo A-I.
• both proteins in the apo E region were identified by immunoblotting with antibody to apo E.
• the molecular weights of the proteins detected by immunoblotting with apo A-I and apo E show a difference of about 4kD (picture not shown).
• the estimated molecular weight of apo A-I is about 27,500 and that of the modified apo A-I is about 31,000.
• SimUarly, the estimated molecular weight of apo E is about 37,000 and that of modified apo E is about 41,000.
• Both apo A-I and apo E are modified by a protein of about 4,000 molecular weight.
• Example 14 Detection of CETP Inhibitor Peptide in Plasma of High HDL, Baboons
• Example 15 Characterization of Poiypeptide by Affinity
• Rabbit antibody was prepared against the 4kD poiypeptide isolated from Hpoproteins obtained from high HDL j baboons. This antibody was used to prepare an immunoarfinity column. The Hpoproteins of d ⁇ 1.21 g/ml were passed over the immunoaffinity column. The bound Hpoproteins eluted with 0.1 M acetic acid and separated in 15% SDS-polyacrylamide reducing gels. 4kD, and 31kD polypeptides, and a minor band corresponding to a 41kD poiypeptide were detected.
• sequence of this poiypeptide was compared to sequence of known proteins using Sequence Data Bank (Reardon, W. R. and Iipman, D.J., PNAS (USA) 85:2444-2448 (1988)), and was found to have 100% homology with human and crab-eating macaque apo C-I.
• the 4kD poiypeptide contained approximately 36 amino adds.
• Three peptides were synthesized beginning from the C- terminal end of the apo C-I sequence. The first peptide contained 9 amino adds, the second peptide contained 21 amino adds and the third peptide contained 36 amino adds.
• the 36 amino acid peptide had an amino add sequence similar to the 4,000 MW poiypeptide, the other two were fragments of the synthetic peptide starting from its C-terminus.
• Antibody against the 36 amino acids inhibitor peptide was prepared in rabbits as described in Example 7, and used for immunoblotting.
• the thus prepared antibody recognized the 4kD peptide as weU as a 31kD poiypeptide from Hpoproteins of high HDL j baboons.
• Hpoproteins from both phenotypes were separated by 10% SDS gel electrophoresis and immuno-blotted. Two protein bands were detected with the antibody by immunoblotting of samples from high HDL j baboons. Only a single band was detected in samples from low HDL j baboons.
• a CE transfer assay as described in Example 4 above was conducted using HDL ! baboon plasma. 3 H HDL and the VLDL+LDL carriers, in the presence of CETP enzyme to mediate the exchange.
• Example 20 Inhibition of CETP From Humans by Synthetic CETP Inhibitor Peptide.
• cholesteryl ester- labeled HDL (10- ⁇ g of cholesteryl esters with a specific activity of 3-4 x 10 6 dpm/mg cholesteryl ester) from low HDL j baboons was incubated with 50-100 ⁇ g of VLDL+LDL cholesteryl ester from baboons.
• the incubations were carried out in the presence of 100 ⁇ l of Hpoprotein defident serum (LPDS) obtained from humans and 2mM DTNB.
• the total volume of the assay was 1 ml.
• the incubations were carried out for 1-2 h at 4° and 37°.
• CETP inhibitor was similar to human apo C-I terminal peptide with 38 amino acids (Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp- Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu- Leu-He-Ser-Arg-Ue-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO. 3).
• Example 21 Method of preparing [ 3 H] HDL Cholesteryl estei labeled HDL, VLDL+LDL LPDS (human).
• VLDL+LDL (d ⁇ 1.045 g/ml) and LPDS were isolated by sequential ultracentrifugation from 100-200 ml blood obtained from two to four
• Experiment 22 Method of isolating human CETP
• blood (5-10 ml) from human subjects was obtained in tubes containing EDTA (1 mg/ml).
• Plasma was obtained by low speed centrifugation. Plasma was kept on ice and the LDL was precipitated by adding 40 ⁇ l heparin/ml (5000 units /ml) of plasma along with 60 ⁇ l/ml of plasma of IM MnCl 2 . The plasma was vortexed and incubated on ice for 15 min. FoUowing incubation, the supernatant was recovered by centrifugation. The procedure was repeated twice to completely precipitate VLDL+LDL in the plasma.
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