JP2003508349A - Methods of treating angina and / or angina-equivalent conditions, and pharmaceutical compositions and kits related thereto - Google Patents

Abstract

  (57) [Summary] The present invention provides a method of treating angina (eg, stable angina, unstable angina and atypical angina) and / or an angina-equivalent condition, wherein the method comprises providing a patient with a therapeutically effective amount for a treatment period. Administering a plurality of liposomes, preferably large liposomes, wherein the liposomes are substantially free of sterols. The method also includes administering an effective amount of an anti-angina drug rather than a liposome. The invention also provides a method of treating lameness, comprising administering a therapeutically effective amount of a liposome. In yet another variation, the invention provides a method of intra- or post-operative conditioning of a subject, comprising administering a liposome. Some other inventions are also described herein.

Description

Detailed Description of the Invention

Patent application to related parties The following is known. The present inventors, Dennis I., who is a US citizen. Goldb
erg (Destination 109 Bent Road, Sudbury, Massachus
setts 01776) and Kevin Jon Willi, a US citizen.
ams (Residence 425 Wister Road, Wynnewood, Penn
Sylvania 19096) invented a new and useful: “angina and / or angina equivalent state (equiva).
Lent) Method of Treatment, and Pharmaceutical Compositions and Kits Associated Therewith "The following is a detailed description of the invention of that specification.

(A method of treatment of angina and / or angina equivalents,
Background of the Invention Chest pain can have many causes (eg, gastric discomfort, pulmonary distress, pulmonary embolism, musculoskeletal pain pneumothorax, non-cardiac coronary conditions, and Ischemic coronary syndrome). Ischemic coronary syndromes include the following: stable angina, unstable angina, variant angina (angina)
, Angina equivalence, myocardial infarction, and related dysfunctions (eg, arrhythmia, low cardiac output, heart failure, infarct dilation, infarct extension, reperfusion injury, and coronary thrombosis) Stable angina is regular and characteristic It is angina that recures in a different pattern. Humans recognize themselves as suffering from angina only after some episodes and patterns have occurred. There is a specific level of activity or stress that stimulates angina, and the pattern generally changes slowly.

Unstable angina is angina that manifests as either a very heavy episode or a frequently recurring angina attack. In unstable angina, the established pattern of angina can change sharply. That is, the angina may appear at rest or may result from far less exercise than in the past.

Atypical angina also refers to Prinzmetal's a angina.
ngina). This differs from the typical angina in that this angina occurs almost exclusively, when the person is at rest. Attacks can be very painful and usually occur between midnight and 8 am. Atypical angina can be associated with acute myocardial infarction, severe cardiac arrhythmias (including ventricular tachycardia, ventricular fibrillation and even sudden death), as well as other forms of angina and angina equivalents. Variant angina results from coronary artery spasm. This spasm can occur near atherosclerotic disorders. Many people with angina experience acute periods of activity. Angina and cardiac events can often occur for 6 months or more.

Angina is defined as chest pain or discomfort of cardiac origin. This usually results from a transient imbalance between myocardial oxygen supply and myocardial oxygen demand. This discomfort is often induced by exercise, emotions, eating, or cold weather. Pain appears to be more likely to occur when the subject is outdoors (especially when the temperature is extremely hot or cold) and when the patient is walking uphill towards a cold. Angina generally refers to when a subject eats heavy metals, or when the subject moves, becomes angry,
Or it happens when you are nervous.

Normal coronary circulation is dominated and controlled by myocardial oxygen demand. This requirement is met by the ability of the heart to significantly fluctuate coronary resistance (and hence blood flow), while the myocardium extracts a high and relatively constant proportion of oxygen (Harrison's Principles of. Internal M
edicine, 12th edition, 1991, Chapter. 16). Normally, resistant arterioles in the myocardium show a vast capacity for dilation. During exercise and emotional stress, changes in oxygen demand affect coronary resistance and thus regulate blood and oxygen supply (metabolic regulation). These same blood vessels adapt to physiological fluctuations in blood pressure to maintain coronary blood flow at a level appropriate to myocardial demand (autoregulation). Although the epicardial coronary arteries can contract and relax, in healthy individuals they act as ducts and "conducting vessels" (cond).
uctance vesels). The intramyocardial arteries, on the other hand, usually show surprising changes in hearing and, therefore, "resistive vessels" (resis
(Tance vesels) (ibid.).

[0007] Once severe stenosis of the proximal epicardial artery reduces cross-sectional area by more than approximately 70%, distal resistant vessels dilate, reducing vascular resistance and coronary blood flow. To maintain. Pressure gradually develops across the proximal stenosis and post-stenotic pressure drops. When the resistant blood vessels are maximally dilated, myocardial blood flow becomes dependent on pressure in the coronary arteries far from the lesion. In these situations, changes in myocardial oxygenation may result from changes in myocardial oxygen demand and changes in the caliber of the stenotic coronary arteries due to physiological basal emotions, pathological convulsions, or small platelet plugs. All these transient events can reverse the important equilibrium between oxygen supply and oxygen reception, and thus lead to myocardial ischemia.

The effects of ischemia are many. Inappropriate oxygenation induced by affective arteriosclerosis can result in transient impairment of myocardial medical, biochemical, and electrical function. Outbreaks of ischemia usually affect the segment of the left ventricular myocardium, with an almost instantaneous dysfunction of normal muscle relaxation and contraction. The relatively poor pericardial perfusion results in more intense ischemia of this part of the wall. Ischemia in a large segment of the ventricles reports transient left ventricular failure. And, when the papillary muscles are involved, mitral regurgitation can compound this event. If the ischemic events are transient, they may be associated with angina; if prolonged, they may give rise to myocardial necrosis and scarring with or without the clinical symptoms of acute myocardial infarction. See: Harrison '
s Principles of Internal Medicine, 12t
h edition, 1991 Chapter. 189.

These underlying mechanistic disorders are a wide variety of abnormalities in cellular metabolism, function and structure. When oxygenated, normal myocardium metabolizes fatty acids and glucose to carbon dioxide and water. In severe hypoxia, fatty acids cannot be oxidized and glucose is broken down into lactate; intracellular pH decreases as myocardium accumulates high-energy phosphatases, adenosine triphosphate (ATP), and creatinine phosphate. To do. Impaired cell membrane function leads to potassium leakage and sodium uptake by cardiac cells. The severity and duration of the imbalance between myocardial oxygen supply and myocardial oxygen demand determines whether the damage is reversible or whether it is homeostatic and subsequently leads to myocardial necrosis. Ha
rrison's Principles of lnternal Medic
ine, 12th edition, 1991.

Ischemia also causes characteristic electrocardiographic changes, such as repolarization abnormalities, as evidenced by T wave inversion and subsequent ST segment displacement (Harrison's Pris).
nciples of Internal Medicine, 12th ed
edition, 1991, Chapter. 176). Transient ST segment suppression often reflects subendocardial ischemia, while transient ST segment elevation is associated with a more severe transmural (tr).
It is considered to be caused by ischemia. Another important consequence of myocardial ischemia is electrical instability. Because it can cause ventricular tachycardia or ventricular fibrillation (Harrison's Principes of Internal).
Medicine, 12th edition, 1991, Chapter. 185
). Most patients who suddenly die from ischemic heart disease die of ischemia-induced malignant ventricular arrhythmias (Harrison's Principles of I).
internal Medicine, 12th edition, 1991, C
hap. 40).

The object of the present invention is to solve the problems associated with conventional therapies used to treat angina, angina equivalents and these various forms of acute coronary syndrome.

SUMMARY OF THE INVENTION The present invention provides a method of treating angina comprising the step of administering to a subject a therapeutically effective amount of liposomes. Stable angina, unstable angina, variant angina, and / or angina equivalents are treated according to the methods described herein. The liposome is selected from the group consisting of large liposomes, small liposomes and combinations thereof, and LDL in the subject.
It is administered so that the levels do not rise substantially.

When large liposomes are used, the large liposomes are the chemical composition of the liposomes in size, function, composition or mode of administration or schedule such that the liposomes do not malignantly impair cholesterol homeostasis. Administering liposomes involves gradually injecting liposomes into a subject in one modification of the invention. In another modification of the invention, small amounts of liposomes are administered over time to avoid elevated LDL concentrations.

The method also includes periodically assaying plasma LDL concentration using an assay that obtains the assayed LDL concentration. This assay comprises a plasma esterified cholesterol assay, a plasma apolipoprotein B assay, a plasma gel filtration assay, a plasma ultracentrifugation assay, and components for determining the level of therapeutically effective amount of each of its liposomes. (Wherein its components are selected from the group consisting of polyanions, divalent cations, and antibodies), plasma ultracentrifugation assays, precipitation assays, immunoturbidimetric assays, and electrophoresis. Is selected from the group consisting of assays.

This method uses a therapeutically effective amount of liposomes in the range of about 10 mg to about 1600 mg NDC / kg body weight / dose. Liposomes are provided periodically during the treatment period in one embodiment. And it is unilamellar liposome, minority layer (
Pauci-lamellar) liposomes and multilamellar liposomes.

Preferably, the liposomes have a diameter greater than about 50 nm, a diameter greater than about 80 nm, and / or a diameter greater than about 100 nm. The liposomes also have a thickness of about 100 nm to about 150 nm, about 150 nm to about 200 nm.
From about 250 nm to about 300 nm, and / or from about 300 nm to about 40
It may have a diameter in the range of 0 nm. Other preferred liposome ranges are described herein.

In another embodiment of the invention, the liposomes are provided to the subject by intravenous bolus administration, intravenous injection, and / or intraperitoneal administration. Other routes are also contemplated (eg, intramuscularly, subcutaneously, intranasally, by inhalation, rectal and by oral or enteral absorption by encapsulation).

The method also includes modifications in which there is monitoring of the cardiac function of the subject. Typical cardiac functions monitored are EKG abnormalities, ST segment changes, arrhythmias,
Compartmental wall assessment, blood viscosity, exercise tolerance, ambulatory EKG monitoring, and / or heart wall motion abnormalities.

In yet another modification of the invention, the method comprises administering an effective amount of an anti-angina drug other than empty liposomes. “Empty” is a standard term indicating the absence of encapsulated drug within liposomes, where the encapsulated drug is essential for one or more functions of the liposome. is there. Includes nitrates, beta blockers, calcium channel antagonists, coronary vasorelaxants, lipid-lowering agents, post-load reducing agents, muscle contractors, pre-load reducing agents, and opiates.

As nitrates, for the purposes of illustration, nitroglycerin, sublingually administered nitroglycerin, long-acting nitrates, sublingual nitrate preparations, buccal (oral) nitrate preparations, oral nitrate preparations, Spray nitrate preparation, oral nitrate spray, isosorbide dinitrate preparation, isosorbide-5-mononitrate preparation, sustained release preparation of isosorbide-5-mononitrate, topical nitroglycerin, nitroglycerin ointment, nitro including transdermal patch Mention may be made of glycerin and nitroglycerin impregnated silicon gels or polymer matrices.

As β-blockers, for the purposes of illustration, non-selective β-blockers, propanol, nadolol, pentabtrol, pindolol, sotalol, timolol, carteolol, drugs that block both β1 and β2 receptors, heart Mention may be made of selective β-blockers, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and drugs that block the β1 receptor with little effect on the β2 receptor.

In one modification of the invention, the calcium channel antagonist is a compound that inhibits calcium antagonists, calcium ion translocation through slow channels in cardiac and smooth muscle membranes by non-competitive blockade of voltage-sensitive L-type calcium channels. , dihydropyridine, nifedipine ^TM, phenylalkyl amines, verapamil ^TM, benzothiazepine, diltiazem ^TM, nicardipine ^TM, amlodipine ^TM, and bepridil ^TM, calcium antagonists of the second generation, Nikarujiopin ^TM, isradipine ^TM, amlodipine ^TM, felodipine ^TM, dihydropyridine It is selected from the group consisting of derivatives.

Yet another aspect of the invention is the step of administering an angiotensin converting enzyme (ACE) inhibitor to a subject in combination with a liposome, administering an antiarrhythmic drug to the subject in combination with the liposome, And / or performing a positive lifestyle in the subject. Typical positive lifestyle changes include weight loss, reduced smoking, loss of smoking, exercise, controlled exercise, decreased salt intake, decreased saturated fatty acid intake, decreased cholesterol intake, and total fat intake. Includes reducing, avoiding physical stress, avoiding emotional stress, and reducing caloric intake.

[0024] Yet a further aspect of the invention involves performing an anti-angina treatment in combination with liposome administration. Anti-angina therapy includes treatment of coexisting exacerbated conditions such as treatment for hypertension, treatment for hyperthyroidism, treatment for lung disease, treatment for heart failure, and treatment for anemia. ..

Another embodiment of the invention involves administering an antithrombotic therapeutic agent in combination with empty liposome administration. The antithrombotic treatment is selected from the group consisting of administering a therapeutically effective amount of an antiplatelet drug, administering a therapeutically effective amount of a drug that inhibits fibrin clot formation, and thrombolytic therapy.

Another embodiment of the invention includes a method of treating claudication. The method involves administering a therapeutically effective amount of liposomes.
As mentioned above, the liposomes are selected from the group consisting of large liposomes, small liposomes, and combinations thereof, and the method can be combined with the steps of the other methods described above and below.

Yet another aspect of the present invention includes a pharmaceutical kit for treating angina comprising: a first container 1 having liposomes and a second container having an anti-angina drug other than liposomes.

The present invention also relates to a method of acclimatizing a preoperative and / or intraoperative condition of a subject. This method involves administering liposomes alone or in combination with administration of anesthetics or sedatives, and / or preoperatively or intraoperatively assessing cardiac function in a subject.

Objects and features of the invention other than those specifically shown above will become apparent in the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION FIG. 1 illustrates a schematic diagram of the structure of normal lipoprotein 100 and unilamellar liposome 200. Lipoprotein 100 and liposome 200 include phospholipid molecule 300. Phospholipid molecules generally have a polar head 500 and a fatty acyl chain 400. Molecule 600 represents a non-esterified cholesterol molecule. The lipoprotein 100 comprises a hydrophobic core 102 composed mainly of triglycerides and cholesterol esters, surrounded by a monolayer of phospholipid molecules 300, whose fatty acyl side chains 400 are towards the hydrophobic core 102, and The polar head 500 is toward the surrounding aqueous environment (not shown). Non-esterified cholesterol 600 is found primarily within phospholipid monolayers. Apolipoprotein 700 is located within phospholipid molecule 300. The artificial triglyceride emulsion particles have essentially the same structure, either with or without protein.

Liposomes 200 contain phospholipid molecules 300 to form phospholipid bilayers (eg, lamella, which may or may not contain proteins, fatty acyl side. The chains 400 face each other, their polar headgroups 500 on the outer leaflets are directed outwards towards the surrounding aqueous environment (not shown), and their polar headgroups 500 on the inner leaflets are aqueous on the particles 200. Inward towards core 202. Depending on the composition of particles 200, the phospholipid bilayer may have a large capacity for non-esterified cholesterol and other exchangeable materials and their components. There is no sterol, as illustrated in Figure 1. Typically, such liposomes are
Unesterified cholesterol can be picked up from other lipid bilayers such as cell membranes and from lipoproteins. Liposomes also pick up proteins and provide them to phospholipids and other exchangeable materials and their components.
Liposomes can also have multiple structures. Here, the bilayer is contained within an environment that is encapsulated by the outer bilayer to form multiple layers. The multiple layers may be arranged concentrically, like layers of onions, or in another modification, non-concentrically.

The liposomes described above are used in a method of treating angina and angina equivalents. Types of angina that can be treated using the method include stable angina, unstable angina, and / or variable angina. In particular, the present invention is effective in treating angina.

The method includes the step of administering to a subject a therapeutically effective amount of a plurality of large liposomes, which comprises a phospholipid substantially free of sterols, to a subject. In one modification of the invention, the large liposomes are fenestratio of liver sinusoids lining the endothelial layer in the liver.
n) of larger size and shape. Preferably, the large liposomes have a diameter greater than about 50 nm, a diameter greater than about 80 nm, or about 100 n.
have a diameter greater than m. Particularly effective liposomes are from about 100 nm to about 20
It has a diameter in the range of 0 nm. In a modification of the invention, the liposomes are selected from the group consisting of large liposomes, small liposomes, and combinations thereof.

A therapeutically effective amount of liposomes is administered from about 10 mg to about 1600 mg phospholipids /
The body weight is in the kg / dose range, and the liposomes are given periodically during the treatment period. Therapeutically effective doses of liposomes may be given in a variety of ways, which illustratively include: intravenous bolus administration, intravenous infusion,
And intraperitoneal administration.

The method of administering liposomes can be combined with the step of determining efficacy of treatment. For example, the method involves monitoring the cardiac function of the subject before, during, or after administration of the liposomes. Pretreatment cardiac function measurements are performed prior to administration of the liposomes. After administration of the liposomes, an improvement in the patient's cardiac function may also be determined to indicate efficacy of the treatment and whether a subsequent treatment is needed, or the time or duration between subsequent treatments. The appropriate dosage for the treatment of can be determined.

There are many cardiac functions that can be analyzed in the present invention. These functions include EKG abnormalities, ST area changes, arrhythmias, evaluation of wall movement, blood viscosity, exercise tolerance, outpatient EKG monitoring, and heart wall movement abnormalities.

Liposomal administration can also be advantageously combined with administration of effective amounts of anti-angina agents other than liposomes, or anti-angina procedures such as LDL phoresis, hematopoiesis, or other revascularization. Anti-angina drugs include, for illustrative purposes, nitrates, beta blockers, calcium channel antagonists, coronary dilators, lipid lowering drugs, post-load reducing agents, muscle contractors, pre-load reducing agents, oxygen, opiates, and their Derivatives and / or combinations are included.It is understood that liposomal administration is also particularly desirable in patients (eg, diabetes) where conventional anti-angina therapies are poorly effective or ineffective.

Exemplary nitrates for use in the present invention include: nitroglycerin, sublingually administered nitroglycerin, long acting nitrates, sublingual nitrate preparations, buccal (oral) nitrate preparations, Oral nitrate preparation, spray nitrate preparation, oral nitrate spray, isosorbide dinitrate preparation, isosorbide-5-mononitrate preparation, isosorbide-5-mononitrate sustained release preparation, topical nitroglycerin, nitroglycerin ointment, via Included are nitroglycerin containing skin patches, and silicon gels or polymer matrices impregnated with nitroglycerin.

Exemplary β-blockers used in the present invention include non-selective β-blockers, propanol, nadolol, pentabtrol, pindolol, sotalol,
Timolol, carteolol, a drug that blocks both β1 and β2 receptors, a cardiac selective β-blocker, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and β2 receptors with little effect on β2 receptors Drugs that can be used.

Exemplary calcium channel antagonists include compounds that inhibit the migration of calcium ions through slow channels in cardiac and smooth muscle membranes by non-competitive blockade of voltage-sensitive L-type calcium channels, dihydropyridine, nifedipine ^™ , phenyl. alkylamine, verapamil ^TM, benzothiazepine, diltiazem ^TM, nicardipine ^TM, amlodipine ^TM, and bepridil ^TM, calcium antagonists of the second generation, Nikarujiopin ^TM, isradipine ^TM, amlodipine ^TM, felodipine ^TM, dihydropyridine derivatives, their combination and their And derivatives thereof.

The present invention may also include administering an angiotensin converting enzyme (ACE) inhibitor, and / or administering an antiarrhythmic drug to a subject to achieve a beneficial result for the subject. Various types of ACE inhibitors are described below. It is also possible to make positive lifestyle changes in the subject to obtain beneficial results. These lifestyle changes include weight loss, reduced smoking, loss of smoking, exercise, controlled exercise, decreased salt intake, decreased saturated fat and saturated fatty acid intake, decreased cholesterol intake, and total fat intake. Decrease,
Avoiding physical stress, avoiding emotional stress, and reducing caloric intake.

Yet another modification of the present invention, in combination with performing anti-angina therapy, comprises a therapeutically effective amount of a plurality of large liposomes (phospholipids substantially free of sterols in the subject during the treatment period). (Eg empty liposomes). Exemplary anti-angina therapies include treatment of concomitant exacerbated conditions. For the purposes of illustration, coexisting exacerbated conditions include, for example, treatment for hypertension, treatment for hyperthyroidism, treatment for lung disease, treatment for heart failure, treatment for anemia, hypermetabolic conditions. And the treatment for diabetes.

Yet another aspect of the invention is to provide a therapeutically effective amount of a plurality of sterol-free phospholipids in the subject during the treatment period in combination with administering an antithrombotic therapeutic agent. Administering large liposomes (eg, empty liposomes). Various types of antithrombotic therapeutic agents are used in the present invention. This includes administering a therapeutically effective amount of an antiplatelet drug (described below), administering a therapeutically effective amount of a drug that inhibits fibrin clot formation, and / or thrombolytic therapy.

The large liposomes described herein (or the small liposomes described herein when administered in a manner such that the deleterious effects of administration on modification are substantially eliminated) are substantially It provides LDL level regulation and / or regulation of other atherogenic lipoproteins. The methods described herein include
It can be combined with monitoring LDL levels in a subject. Monitoring LDL levels in the subject when the liposome is a modification selected from the group consisting of large liposomes, small liposomes and / or combinations thereof,
And more importantly, it may be desirable to administer the small liposomes in such a way that LDL levels in the subject are not substantially elevated. This can be by injecting liposomes slowly and / or by administering small doses of liposomes at separate times to avoid elevated levels of lDL, or by administration of cholesterol-lowering agents or co-administration of large liposomes. Can be achieved by

To monitor LDL concentration, plasma LDL concentration is regularly assayed using an assay that obtains the assayed LDL concentration. This assay
Plasma esterified cholesterol assay, plasma apolipoprotein B assay, plasma gel filtration assay, plasma ultracentrifugation assay, and component (eg, this component comprises a polyanion, a divalent cation, and an antibody. Selected)). Other assays used herein include determining the level of therapeutically effective amount of each of the liposomes,
Plasma ultracentrifugation assay, sedimentation assay, immunoturbidimetric assay, and / or
Or an electrophoretic assay is mentioned.

It is also understood that the other method steps described above can be utilized with combinations of liposomes of different sizes, and that a particularly useful type of liposome comprises POPC. Liposomes containing phospholipids that are liquid crystals at body temperature but are still resistant to oxidation are desired. It is understood that liposomes transport cholesterol and other exchangeable materials and donate phospholipids, in part, in association with HDL and with transfer proteins such as phospholipid transfer proteins and cholesteryl ester transfer proteins. It These can be endogenous or exogenous.

In another aspect of the invention, pharmaceutical kits for treating angina are also described herein. The kit comprises a first container having liposomes; and a second container having an anti-angina drug other than liposomes. Exemplary anti-angina drugs are described above.

Yet another particular useful aspect of the invention involves a method of preoperative or intraoperative habituation of a subject, which method comprises administering liposomes. Many subjects are at risk of complications when undergoing surgery. Many of these risks can be reduced by administering certain effective, empirically determined institutional liposomes prior to surgical procedures. Here, the patient's body is at risk of stress. The method of preoperative acclimation of the subject can be combined with treatments such that the risk of patient complications and / or stress associated with other known preoperative steps and surgical procedures can be minimized. These other pre-operative steps include, for illustrative purposes, administration of anesthetics or sedatives,
Examples include preoperative evaluation of the subject's heart function.

The liposomes of the present invention are advantageously and efficiently manufactured using the novel methods and systems described herein. A desired substantially uniform size range (eg,
Generating liposomes of 125 nm to 250 nm) is particularly slow using conventional methods. This is because during the initial production, the first pass of the liposome / liposome precursor pair is difficult, since large particles pass through the glacier slowly in conventional extrusion devices. is there.

Accordingly, the present invention includes a commercial process for producing large quantities of liposomes within the desired size range. The method involves hydrating a phospholipid. This hydration step initially produces large particles that are difficult to pass through an extruder with a pore size in the desired range. Thus, this method utilizes high throughput equipment to bring the particles into a size range that readily passes through an extruder with a filter to produce the desired particle size (about 200 nm to about 400 nm). Pre-conditioning the hydrated liposomes by means of extruding the pre-conditioned particles subsequently with a filter of the desired size (eg size of about 100 nm or other depletion size). The step of passing through a vessel. Exemplary high-throughput devices include microfluidizers, homogenizers, shear-based devices, and / or extruders with large pore sizes (500 nm or other suitable large pore sizes). It will be appreciated that the above method significantly reduces the manufacturing and / or processing time to obtain liposomes in the desired size range. The production rate bottleneck problem is eliminated by the preconditioning step described above.

There are populations of LDL that are considered to be particularly harmful (eg, the pattern βL
Low density LDL also known as DL). Pattern βLDL is commonly found in diabetes, hypertriglyceridemia, low HDL status, hypertension, insulin resistance, hyperinsulinemia and / or Syndrome X status. Low density LDL is characterized by poor lipids, especially with respect to surface lipids, including non-esterified cholesterol and phospholipids.
LDL can acquire phospholipids from liposomes. Treatment of the subject with phospholipid liposomes enriched for low density LDL and restored it to normal phospholipid content,
Reduce its atherogenicity and / or normalize its carbohydrate content and / or its size. Treatment with the compositions described herein includes
It alters the surface charge density of low density LDL and brings it to normal surface charge density. Lipoproteins in subjects with low density LDL show enhanced binding to proteoglycans (the strongest binding of that lipoprotein is IDL). The compositions and treatments described herein improve the deleterious properties of low density LDL and other lipoproteins commonly found in subjects with low density LDL.

Examples of deleterious properties ameliorated by the treatments disclosed herein include strong binding to the arterial matrix, easy oxidizability, ease of penetration into the arterial wall, and LDL receptors. Includes, but is not limited to, binding disorders.

The present invention further provides methods of treating the accelerated syndrome of vascular dysfunction.
Accelerated syndromes of vascular dysfunction include renal disease, transplanted atherosclerosis,
Asymptomatic ischemia, and its manifestations on all body parts (particularly brain, legs, genitals, and heart), ischemia, vascular grafts, diabetes mellitus, diabetes mellitus with nephropathy, diabetes mellitus with proteinuria Is included. The present invention also provides methods of treating other perfusion abnormalities such as hyperviscosity syndrome, sickle cell dyskeratosis, conditions involving impaired wound healing, and / or conditions involving aggregation of blood cells or platelets.

Organ perfusion can be determined herein by tests such as PET scans, Doppler evaluation, evaluation using contrast agents, and pulse wave examination.

Post-load reducing agents as used herein include antihypertensive agents and peripheral vasodilators (eg nitrate) and calcium channel antagonists.

The present invention also relates to male and / or female sexual dysfunction, acute coronary syndrome,
From stable coronary syndrome, intermittent claudication, transient ischemic attack, stroke, heart attack, myocardial infarction, myocardial ischemia, ischemia to any body part, organ failure from ischemia, and / or ischemia For treatment of organ infarction.

Administration of liposomes promotes angiogenesis, eg, formation of collateral vessels.
Lateral visual angiogenesis is a disorder in hypercholesterolemia and is affected by related particles containing LDL or apo-B. Therefore, the use of this method is a beneficial treatment for subjects who can benefit from accelerated angiogenesis, including collateral angiogenesis.

3 and 4 illustrate plasma LDL cholesterol ester concentration over time for LUV, SUV, or saline infusion. Rabbit with arrow 302
, 304, and 306, respectively, at days 1, 3, and 5 injected intravenously with a bolus of phosphatidylcholine 300 mg / kg body weight or a matching volume of saline. Its phosphatidylcholine, about 100 nm (preferably, about 120 nm) having a diameter of, large unilamellar particles (LUV) (LUV prepared by extrusion method, about the Nicomp ^TM model 370 submicron laser particle size determination apparatus 120 nm ( 123 ± 35 NM), and the extruded membrane had pores with a diameter of about 100 NM), or a small single particle with an ultrasonically prepared diameter of about 30 NM (preferably 35 NM). It was a pharmaceutical grade egg PC in either form of layered particles (SUV measured in the range 34 ± 30 NM). Blood was collected before each injection and at day 6 sacrifice. Plasma LDL cholesterol ester concentration was determined by plasma gel filtration assay by in-line enzyme assay for cholesterol ester. Mean ± SEM is shown in FIG. 3 animals injected with SUV
At days 5, and 6, there was significantly higher plasma concentration of LDL cholesterol ester compared to either LUV-injected or saline-injected animals. 2-8
, 10-15, 24, and 28 exemplify data from the same experiment in which infusions were made on days 1, 3, 5 and then the liver was removed. Gel filtration was performed on plasma to determine the lipid content of individual liposome classes. FIG. 2 shows C
ETP, HMG-CoAR (hydroxymethylglutaryl coenzyme A reductase), LDL receptor, and cholesterol 7-α-hydroxylase; and saline (HEPE for the experiments described with respect to FIGS. 3 and 4).
S-buffered saline) (rabbits 1-4), LUV (rabbits 5-8), and SU
Liver mRNA content for LDL ChE (low density lipoprotein cholesterol ester) for rabbits fed V (rabbits 10-12) (pg /
μg) table is illustrated. Rabbit 13 is a "Mix" rabbit.

FIG. 4 shows animals labeled Mix. "Mix" means the first day, the third day,
And a single animal that underwent SUV on day 5 and also received a single injection of LUV on day 3. Prior to this injection of LUV, the plasma concentration of LDL cholesterol ester was elevated, but after injection of LUV, the LDL concentration was decreased despite continuous administration of SUV.

FIG. 5 shows that, in response to injection of LUV, SUV, or saline over time,
3 illustrates LDL receptor mRNA levels in liver. The rabbits were sacrificed on day 6 and liver samples snap-stored in liquid nitrogen, mRNA was extracted and rabbit mRNA for the LDL receptor was quantified by an internal standard / RNase protection assay (Rea. T. J. et al., J
． Lipid Research 34: 1901-1910, 1993 and Pap. E. , Genet. Anal. 8: 206-312, 1991).
. Mean ± SEM is shown in FIG. Animals injected with SUVs showed significant suppression of liver LDL receptor mRNA compared to animals injected with LUVs or saline. Inhibition of liver LDL receptor mRNA reflects parenchymal cells overloaded with sterols and is a potentially deleterious change from normal liver cholesterol homeostasis. In contrast, LUV-injected animals show the highest levels of hepatic LDL receptor mRNA, but the increase over that observed in their saline-injected animals is not statistically significant. There wasn't. Liver from the above "Mix" animals was 5.28 pg LDL receptor mRNA /
Values in micrograms are given. This is closer to the mean value in the saline group than in the SUV group. Therefore, LDL receptor mRNA was stimulated by a single injection of LUV despite repeated injections of SUV.

FIG. 6 illustrates HMG-CoA reductase mRNA levels in the liver in response to LUV, SUV, or saline injection. This experiment is above. Animals injected with SUVs showed significant suppression of liver HMG-CoA reductase mRNA compared to animals injected with LUVs or saline. Suppression of liver HMG-CoA reductase mRNA reflects parenchymal cells overloaded with sterols, which may be a potentially deleterious change from normal liver cholesterol homeostasis. In contrast, LUV-injected animals showed the highest levels of liver HMG-CoA reductase mRNA, but
The increase over that observed in saline infused animals was not statistically significant.

“Mix” animals had 0.50 pg HMG-CoA reductase mRNA /
The microgram value is shown, which is the mean value (0.51) in the saline group.
, And is substantially higher than the value in the SUV group (0.27). Therefore, HMG-CoA reductase mRNA was stimulated to its normal value by a single injection of LUV, despite repeated injections of SUV.

FIG. 7 illustrates cholesterol ester transfer protein mRNA levels in the liver in response to LUV, SUV, or saline injection. The details of the experiment are referred to above. Animals infused with LUV compared to animals infused with SUV or saline with liver C
It showed significant suppression of ETP mRNA. Suppression of CETP mRNA resulted in alterations in plasma lipoprotein profile normally associated with reduced risk of atherosclerosis. "Mix" animals are 3.18 pg CETP
The value of mRNA / microgram was shown. This is closer to the mean value in the LUV group than in the SUV or saline groups. Therefore, CETP
mRNA was suppressed by a single injection of LUV despite repeated administration of SUV.

FIG. 8 illustrates cholesterol 7α hydroxylase mRNA levels in the liver in response to LUV, SUV, or saline injection. Details of the experiment are as described above. SUV-injected animals showed liver 7α-hydroxylase mRNA compared to LUV-injected or saline-injected animals.
Showed suppression of. Inhibition of 7α-hydroxylase can be a potentially deleterious change from normal liver homeostasis. In contrast, LUV-injected animals
Although it showed the highest levels of liver 7α-hydroxylase mRNA, the increase over that observed in saline infused animals was not statistically significant. “Mix” animals had 0.51 pg of 7α-hydroxylase mRNA
/ Microgram value was shown, which was higher than the mean value in the SUV group. Thus, 7α-hydroxylase mRNA was stimulated by a single LUV injection despite repeated SUV injections.

FIG. 10 shows non-esterified cholesterol levels in whole plasma over time in response to LUV, SUV, or saline. Details of the experiment,
As above. As shown in this figure, LUV and SUV significantly increased plasma concentrations of non-esterified cholesterol. This indicates immobilization of the tissue store. LUVs increased non-esterified cholesterol levels more than SUVs.

FIG. 11 shows the esterified cholesterol concentration in whole plasma over time in response to LUV, SUV, or saline. Details of the experiment are as described above. SUVs increased plasma levels of cholesterol ester on days 3, 5, and 6. FIG. 12 is a copy of the information contained in FIG.

FIG. 13 shows plasma VL in response to injection of LUV, SUV, or saline.
The DL esterified cholesterol concentration is shown. SUV is saline or LUV
Plasma concentrations of VLDL cholesterol ester were increased above those observed in the treatment group. "Mix" animals received 2.4 mg / dl plasma V on day 6.
The LDL cholesterol ester concentration was shown. This is lower than the average value in the SUV group. Details of the experiment are as described above.

14 and 15 showed HDL esterified cholesterol levels in response to LUV, SUV, or saline injections. Details of the experiment are as shown in FIG. Suitable phospholipids are Avanti Polar Lipids, Ni
popon Oil and Fat in Japan, and Princet
on Lipids, as well as other suppliers. LUV is
It is made by a commercially available extruder. SUVs caused a small, but statistically significant increase in HDL cholesterol ester concentration on days 5 and 6.

FIG. 16 shows control or Jacjson Laboratories,
apoE KO (commercially available from In Bar Harbor, Maine)
Knockout) shows the time course of cholesterol immobilization after LUV injection in mice. Control (C57 / BL6) and apolipoprotein E knockout mice were infused with a single bolus of 300 mg LUV phospholipids / kg body weight at time zero. The LUV contained traces of labeled cholesterol hexadecyl ether. It remained on the liposome after injection into mice. The data presented are for total cholesterol concentration in total plasma (ie esterified + non-esterified). The rise in both sets of animals is
We show that LUVs immobilize cholesterol in plasma even in the presence of severe genetic hyperlipidemia.

FIG. 17 illustrates the time course of LUV clearance in control and apoE mice. Details of this experiment are as described in FIG.
Clearance of plasma-derived LUVs was not diminished in apoE knockout mice, which indicates that cholesterol mobilization (FIG. 16) and placement (FIG. 17) even in the presence of severe genetic hyperlipidemia. ) Is shown. This shows the utility of this preparation in hyperlipidemia.

FIG. 18 illustrates another application for the compositions and methods of the invention in humans.

FIG. 19 illustrates a perspective view of the improved hemodialysis system and improved method of hemodialysis of the present invention. Among other benefits, this improved method allows for the treatment of angina, angina equivalent conditions, lameness, and related conditions, and allows pre-operative conditioning during or near the time of dialysis. . Blood was drawn from a site for circulatory access (shown here as arm 1900), and cells-
Transported to plasma separator 1910. This plasma is then transferred to the dialysis chamber 192
It is separated into at least two components that are transported to zero and separated by a semipermeable membrane 1930. One side of the membrane 1930 is patient plasma 1940 and the other side is dialysate 1950. The selected molecular exchange passes through the membrane 1930 depending on the characteristics of the membrane (charge, pore size, etc.). Device 1960 includes devices for adding a lipid acceptor to the dialysate and for sampling the dialysate, including cholesterol, phospholipids, and other components (eg, acceptors, specific lipoproteins, specific components). ) Assay and monitor placement. Extraction of plasma cholesterol or other extractables includes several possibilities: 1) the acceptor is located in the dialysate that does not pass from the membrane 1930 to the plasma; 2) the acceptor is the membrane 1930. It either passes and is returned to the patient while still in plasma or separated from the plasma and then returned to the patient. And / or 3) a sheet (eg, membrane 130)
The acceptor immobilized on (on itself), on the beads, and / or on the walls of the chamber 1920. The plasma thus treated is usually returned to the patient after being remixed with blood cells. As shown, the cholesterol acceptor can be added at any stage as in the example, device 1970 includes the acceptor and simply adds the acceptor to plasma. The return of plasma to the patient then also
It is illustrated in FIG. It is further understood that contaminating cellular material in plasma (eg, platelets) is also cholesterol depleted and phospholipid enriched in endogenous lipids. All acceptors mentioned throughout this application can accept molecules in addition to cholesterol and donate substances as well.

The cell-plasma separator 1910-derived cell concentrate may then be treated in any of several ways and then returned to this patient: 1) returned to the patient without further treatment ( This involves mixing with plasma treated as above); 2) this dialysate containing cholesterol acceptors for lipids,
The cells are transported to a second dialysis chamber (not shown), which depletes the cells' endogenous lipids (eg, cholesterol), after which they are returned to the patient; 3) a suspension or solution of a lipid acceptor for the lipids. Mixed, depleting the cells of their endogenous lipids and then returning to the patient with the acceptor, or with further separation into specific cell types, or after this separation, option 1) and option 2) above. It can either be performed in all cell types or reverted (eg, purified platelets can be lipids depleted of endogenous lipids (eg, cholesterol) and enriched in liposomal lipids). . Options 2) and 3) are
Cellular cholesterol, phospholipids, flux, viscosity, fragility, cell composition and / or
Alternatively, periodic assays of cell function may be performed. Devices 1960, 1970
Includes devices that allow for periodic sampling of cells during treatment. Similar to plasma, lipid acceptors can be added at any stage of treatment. All fluids (
(Eg plasma and concentrated cells), specific gravity, mechanical, manually operated (syringe)
On or with a pump if necessary. Of course, it is understood that blood can be drawn for processing from any suitable part of the body.

FIG. 20 illustrates a perspective view of an improved peritoneal dialysis system 2000 and peritoneal dialysis method. Among other benefits, this improved method allows for the treatment of angina, angina equivalent conditions, lameness, and related conditions, and allows pre-operative conditioning during or near the time of dialysis. . Patient's abdomen 2010 (Fig. 20-
21) receives the peritoneal dialysate 2020 stored in the container 2030 into the peritoneal cavity via the channel 2050 and the incision 2040. The lipid acceptor and / or cholesterol acceptor 2060 may be added to the container 20 as needed.
70. In another refinement, the lipid acceptor is added to the dialysate 2020; simply added to the container 2030 in a concentrated form prior to infusion; added to the fluid stream entering the peritoneal cavity as shown; or Injected by a separate entrance to the patient by any effective route. Throughout this application all acceptors can accept molecules in addition to cholesterol and donate substances such as phospholipids and antioxidants.

FIG. 21 illustrates a perspective view of various improved peritoneal dialysis systems 2100 with assay means and methods of peritoneal dialysis and analysis of waste fluid. Vessel 2110 receives waste fluid from abdomen 2010 via channel 2120. Device 2110 is
It provides access to a diagnostic sample of waste dialysate and allows the assay of cholesterol, phospholipids, and other parameters described herein to demonstrate the efficacy of the described treatments. If desired, assay syringe 2130 is inserted into channel or tube 2120, or container 2110 via access inlet 2140. An optional pump (not shown) is then used to move the various fluids to the appropriate location in the assay.

FIG. 22 is a perspective view of an improved cardiac catheterization and / or angioplasty system 2200 and methods of cardiac catheterization and / or angioplasty in the context of angina or angina equivalent or associated ischemic injury. Is illustrated. Patient 2210 undergoes cardiac catheterization and / or angioplasty. This patient receives an effective dose of lipid or cholesterol acceptor 2230 intravenously co-administered with the treatment from container 2220. Intra-arterial access of catheters for coronary angiography and / or angioplasty allows preparation of co-administration of cholesterol acceptors and of diagnostic agents such as cholinergic agents to assess vascular function To

FIG. 23 shows a perspective view of various improved cardiac catheterization and / or angioplasty systems 2300 and cardiac catheterization and / or angioplasty in the context of angina, angina equivalent or other ischemic disorders. The figure is illustrated. The catheterization and / or angioplasty catheter 2310 has a device 2320 that allows the cholesterol acceptor to exit therefrom. In a modification, the catheter 2310 has a permeable membrane. This membrane allows the cholesterol acceptor to exit from it. Pseudo arrow 2330 indicates the exit site of the cholesterol acceptor and / or diagnostic agent. Site 2340 represents the acceptor or drug entry site. The balloons on device 2300 may be replaced or supplemented with other devices, or they may form an inner balloon layer located within an outer balloon layer. The acceptor is located between the inner flexible balloon layer and the outer flexible balloon layer. Upon inflation of the inner balloon layer, a force is exerted against the fluid or gel-like acceptor that forces the acceptor outside site 2320, and to a more localized direct treatment of arterial trauma ( Strongly)
Contact directly. It will be appreciated that various inventions provide maximum penetration of the acceptor at the site of the artery. This infusion can be accomplished by specific gravity, manual operation of the syringe, or by a mechanical infusion pump 2350. Similar methods and systems are
It can be utilized in standard vascular imaging techniques or in vessels including the femoral, carotid, and mesenteric vessels as an example.

Patient 2210 undergoes cardiac catheterization and / or angioplasty. The patient receives an effective dose of cholesterol or lipid acceptor 2230 intravenously co-administered with the treatment from container 2220. Intra-arterial access for coronary angiography and / or angioplasty, such as cholinergic agents for preparing co-administration of lipid or cholesterol acceptors and for assessing vascular function and / or organ perfusion Allows preparation for administration of diagnostic agents.

Container 2110 receives waste fluid from abdomen 2010 via channel 2120. Device 2110 provides access to a diagnostic sample of spent dialysate, which allows assaying of cholesterol, phospholipids, and other parameters described herein to demonstrate efficacy of the described treatments. If desired, assay syringe 2130 is inserted into channel or tube 2120 via access inlet 2140. An optional pump (not shown) is then used to move the various fluids to the appropriate location in the assay.

FIG. 24 illustrates a graph of hepatic fluid content against LUV, SUV, or saline infusion. Details of the experiment are as outlined above. Liver samples were analyzed for some lipids (cholesterol ester (CE); triglycerides (TG).
); Non-esterified cholesterol (Chol); phosphatidylethanolamine (PE); and phosphatidylcholine (PC)) were assayed (these lipids are shown in units of μg (microgram) lipid / mg).
Low values of PE and PC were produced in SUV-treated animals; therefore, the Chol: phospholipid ratio in these animals was higher than in the other groups.

FIG. 25 illustrates cholesterol ester concentrations after repeated injections of SUV or LUV (300 mg / kg) in NZW rabbits (New Zealand white rabbits). Arrows indicate the time of phospholipid injection on days 0, 3 and 5. For a given phospholipid dose, LUVs promote a large increase in free cholesterol levels in plasma.

FIG. 26 shows SUV or L in NZW rabbits in the same experiment as FIG.
3 illustrates the free cholesterol concentration in plasma after repeated injections of UV (300 mg / kg). Arrows indicate the time of injection of phospholipids. L, not SUV
Repeated injections of UV did not induce a dramatic increase in plasma CE concentration.

Elevated plasma CE concentration due to the delivery of excess cholesterol to the liver can be a continuous two process. This is due to overproduction of CE-enriched particles or CE
It may be involved in impaired clearance of enriched lipoproteins. The overproduction of CE-enriched particles that occurs after SUV injection can occur in plasma or in the liver. In plasma, LCAT acts on small unilamellar phospholipid vesicles (with empty or encapsulated drug) or on phospholipid-enriched HDL-producing CE, followed by CETP on LDL. Can be shipped to. Results using gel filtration of plasma from SUV-treated animals indicate that CE is transported mostly or substantially on LDL. In plasma, VL by SUV
Removal of apoE from DL slows the clearance of VLDL, thereby helping to more efficiently convert to LDL. In the liver, elevated delivery of cholesterol to hepatocytes during the recruitment of cholesterol from surrounding tissues has been described by apoB.
Stimulates hypersecretion of CE-enriched lipoproteins.

In the modification, the observed elevated plasma CE concentration is the result of defective clearance of CE-enriched atherogenic lipoproteins. Intravenously administered liposomes that acquire apoE compete with LDL for LDL-receptor mediated uptake. Delivery of excess cholesterol to specific regulatory pools in the liver
Downregulates the L receptor, which interferes with hepatic uptake of atherogenic lipoproteins from plasma. For example, LDL removal from plasma is reduced; VLDL removal is also impaired, favoring its conversion to IDL and LDL. The processes responsible for increasing plasma CE concentrations differ between the two liposomal preparations. Unlike SUV, LUV does not induce an increase in plasma CE concentration. LUV is
It is an excellent preparation that mobilizes tissue cholesterol and other exchangeable substances but does not cause harmful side effects.

The methods and compositions of the present invention also provide for enrichment of HDL cholesterol ester by SUV. One contributing process is the stimulation of lecithin cholesterol acyltransferase (LCAT), and other processes associated therewith. The ability of SUVs to increase HDL cholesterol ester is LCAT
Is the result of stimulation and other processes associated with it. LCAT requires phospholipids and cholesterol to produce cholesterol esters and lysophosphatidylcholine; liposomes can supply excess phospholipids.
The invention also relates to changes in lipoprotein (LDL, HDL, etc.) composition,
And LUV and / or SUV and / or other acceptor modifications in function.

The liposome compositions described herein and methods of utilizing the liposome compositions also include liposomes that take up endogenous apoE and thus block the cellular uptake of LDL. Liposomes take up apolipoproteins such as apoE and apoA-I, which alter or enhance their function. For example, uptake of endogenous apoA-I enhances the ability of liposome-derived phospholipids to take up cholesterol. And endogenous ap
Uptake of oE allows liposomes to block specific pathways for arterial uptake of lipoproteins. All of these are in the context of regulation of LDL levels, and hepatic gene expression, and cholesterol homeostasis.

LUVs and SUVs deliver cholesterol to different regulatory pools within the liver. This conclusion indicates that differences in hepatic gene responses and CETP mRNA are suppressed; LDL receptor mRNA is unaffected or increased by LUV but suppressed by SUV; and CETP is suppressed by LUV. , Supported by being unaffected by SUVs.

Important points regarding LUVs are illustrated in FIG. The practical advantage of using LUVs as a treatment for angina, angina equivalents and other ischemic disorders is that they are simple to manufacture, allergen free, synthetic and at very high doses. Even is non-toxic. Mechanically, LUV is LD
It promotes reverse cholesterol transport in vivo without inducing elevated L levels, and LUV is the optimal preparation. These particles are often referred to as "empty" vesicles, but drug uptake into the aqueous interior or bilayer of phospholipid vesicles also disrupts at least one essential action of the vesicle. It is understood that it can be implemented without.

The compositions used herein can be cleared and eliminated directly from hepatocytes. And the various methods described herein are utilized with the slow composition infusions described, so that hepatocytes are not overloaded with cholesterol, even if clearance by parenchymal cells occurs. Moreover, HDL is also regulated by CETP gene repression.

The assay is performed as described herein by: VLD
Assaying fasting plasma triglycerides to estimate L concentration; assaying plasma cholesterol (free ester, or total-free ester); precipitating LDL (and VLDL) with polyanion-cation; H
Assaying the supernatant, which is DL; and calculating the (total plasma value-VLDL-HDL) sterol (or sterol ester) of LDL in total plasma. Liposomes are precipitated with polyanion-cations; or, if desired, esters lacking most liposomes are assayed. Other assays include electrophoresis, chromatography, immunoassays, electron microscopy assays, functional assays, structural assays, and compositional assays.

In the dialysate of the invention, any liposome or emulsion can be used as long as they are cholesterol acceptors and they do not elevate LDL or they are returned to the circulation of the patient. In either case, it is necessary to assay plasma LDL and acceptor plasma concentrations, as well as other atherogenic lipoproteins plasma concentrations.

For methods requiring delivery of cholesterol to the liver at a slow rate or low dose, the administration is a small acceptor (eg, SUV) without LUV.
Can be used, except that LDL levels are monitored and regulated as levels of other atherogenic lipoproteins. To avoid disrupting hepatic cholesterol homeostasis, the drug entrapped as described herein need not be given in low doses, and the encapsulating liposomes or emulsions should be given in low doses. The drug may be abundant in a few liposomes or a small amount of liposomal lipids.

Alterations in the size, composition, and function of HDL can be achieved by administering high or even low doses of large and / or small liposomes with little or no sterol. Liposomes without sterols are readily split into HDL and HDL apolipoproteins when given at low doses, and then the pieces are enriched in plasma HDL within phospholipids.
Incorporated into the DL fraction. For lower LDL levels, even LUVs or even drug-free SUVs are at such low doses (eg 10-100 mg).
/ Kg / dose) is unlikely to increase plasma LDL levels, but periodic monitoring is advisable.

The method disclosed herein, which alters LDL composition without increasing LDL concentration, also uses a phospholipid such as POPC (palmitoyl oleoylphosphatidylcholine) that is resistant to oxidation. Enrich the composition, enrich it with antioxidants, deplete non-esterified cholesterol, and reduce cellular or arterial uptake of oxidized LDL by phospholipid enrichment.

Liposomes up to about 1000 NM are utilized in the present invention. Larger amounts of liposomes are also utilized, but extraction of tissue lipoproteins can be inefficient. Further, the composition of the present invention can be concentrated or dried. These preparations are then diluted or reconstituted at the time of treatment or administration.
In this variant, a two-component kit containing the active substance and a diluent is provided. Encapsulation of phosphatidylglycerol (PG) to create negatively charged liposomes or other charged components in the composition to avoid aggregation during storage is also
Provided.

FIG. 27 illustrates changes in plasma components after repeated injections of SUV. Watanabe hereditary hyperlipidemia (WHHL) rabbits receive 1000 mg / kg body weight of SUV phospholipids or an equivalent amount of saline intravenously over 3 weeks each Monday, Wednesday, and Friday (9 total doses). Gave to. Three days after the final dosing, blood samples were taken and plasma components were fractionated by size by passing through a Superose-6 gel filtration column. The eluate was read by an in-line spectrophotometer. The traces on the right are from saline injected rabbits and show approximately VLDL in fractions # 17-18, and approximately LDL in fraction # 27. The trace on the left is from a SUV-injected rabbit and shows approximately VLDL with persistent liposomes in fraction # 16, and approximately fraction # 25 LDL sized particles. This tracking is
Consistent with the increase in LDL, which is a detrimental effect, shows an increase in the amount of LDL sized particles following repeated injections of SUV. Since WHHL rabbits have a genetic defect in the LDL receptor, this result is not only due to SUVs suppressing the LDL receptor (FIG. 5) but also by a mechanism independent of the LDL receptor (FIG. 27). It is shown to destroy hepatic cholesterol homeostasis. LUV avoids both LDL receptor-dependent and LDL receptor-independent destruction.

FIG. 28 shows agarose gel electrophoresis of whole plasma after repeated injections of LUV, SUV, or saline. Experimental details are cited by reference elsewhere in Figures 2-8 and elsewhere herein. 4 μL plasma samples from 2 rabbits in each group on day 6 were electrophoresed through 1% agarose and then stained with Sudan Black (O: origin, β: LDL standard migration). The SUV-mediated increase in LDL concentration is shown in these lanes by a darker, but otherwise unnoticeable β band. SUVs in plasma showed a mobility superior to LDL. This is mainly due to the acquisition of plasma proteins from HDL.
In contrast, plasma LUVs exhibit essentially the same mobility as freshly prepared protein-free vesicles (ie, just above the origin (O)), which is a substance parenchyma of the proteins acquired in LUVs. Absent or decreased in general.

Based on the electrophoretic mobility of FIG. 28, a quantification of protein acquisition by LUV versus SUV is obtained. LUVs and SUVs were incubated in vitro with human HDL at 37 ° C. for 4 hours, then separated from HDL by gel filtration chromatography and assayed for proteins and phospholipids. LUVs acquired 1.09 μg protein / mg liposomal phospholipids, while SUVs acquired 40.4 μg / mg (ie almost 40-fold). Therefore, the two types of liposomes show significant quantitative differences in protein adsorption. SUVs, but not LUVs, eagerly deprive VLDL of apoE, thereby slowing its clearance from plasma and favoring its conversion to LDL. Furthermore, adsorbed proteins play a role in directing SUVs into hepatic metabolic pools that disrupt hepatic cholesterol homeostasis, whereas LUVs do not direct such pools. Liposomes, emulsions, or any other particles or compounds that extract tissue lipids but do not acquire large amounts of plasma proteins behave like LUVs in these respects.

Specific vascular genes affected by cellular cholesterol loading include prolyl-4-hydroxylase; hnRNP-K; osteopontin (a role for oxidized lipids in inducing intra-arterial calcification). ); And the gene for Mac-2. The methods of modulating these genes described herein result in restoration of normal vascular or arterial function. Elevated expression of prolyl-4-hydroxylase (an enzyme in collagen synthesis, a component of fibrotic plaque) and hnRNP-K (identified in pre-mRNA metabolism and cell cycle progression) message is associated with aortic smooth muscle after cholesterol feeding. Found in cells. These normalize after liposome treatment as described herein. Other genes or enzymes that become abnormal with cholesterol loading are normalized by liposome treatment as described herein (osteopontin, nitric oxide synthase (NOS), adhesion molecules, chemoattractants, Tissue factor, PAI-1
(Plasminogen activator inhibitor), tPA (tissue plasminogen activator), and Mac-2 (Ramaley et al., 1995)).
Other genes affected by cholesterol, cholesterol load, oxidized lipids are also corrected.

Many examples of small acceptors, such as SUVs, apolipoprotein-phospholipid disks, and HDLs are commercially available and can be used in the present invention. Kilsdonk EP et al., Cellular cho
lesterol affiliated by cyclodex
trins, J. Biol. Chem. 270: 17250-17256, 19
95. As a further example, another small acceptor is cyclodextrin. Small acceptors (especially HDL) shuttle cholesterol from cells to liposomes. Cyclodextrins and also other small acceptors can shuttle cholesterol or other exchangeable substances from cultured cells to the LUVs, which substantially removes and donates the substances between the cells and the LUVs. To increase. Apolipoprotein-phospholipid discs include, by way of example, wild-type apoprotein, apoprotein variants, apoprotein variants, apoprotein derivatives, and recombinant apoproteins.

Examples of antihyperlipidemic agents include fibric acid derivatives, HmG CoA reductase inhibitors, niacin, probucol,
Bile acid binders, other agents and combinations thereof. Antihyperlipidemic treatments also include LDL apheresis, ileal bypass, liver transplantation and gene therapy.

The data presented in this application support these possible tests for differences in metabolic response to LUV vs. SUV. The three mechanisms work either separately or in combination. First, LUVs are predominantly utilized by Kupffer cells, while SUVs are predominantly oriented in the hepatic parenchyma. This is a result of a partial mechanism of hepatic architecture: the hepatic endothelium window is an oval-shaped opening of approximately 100 × 115 nm, through which SUVs of 30 nm diameter pass or are easily passed. Get and access the real. Large particles (eg large liposomes of sufficient diameter) do not readily pass through and are instead cleared by macrophage Kupffer cells that line the sinusoids of the liver. SUVs are also accessible to Kupffer cells, but their sheer number (about 10 times the same SUV as LUV / mg of phospholipids) appears to saturate the reticuloendothelial system, and this substance Other methods of orienting artificial particles away from the parenchyma also include, for example, altering particle structure or composition (specific for charge and cell-specific binding). (Including a ligand).

The cholesterol gap pathway mediated by parenchymal versus Kupffer cells has different metabolic consequences. Direct delivery of cholesterol to the parenchyma by SUV suppresses sterol-responsive messages (FIGS. 5, 6 and 8). After delivery of cholesterol to Kupffer cells, for example, spreading of Kupffer cells (Dis
It is followed by a progressive transfer of lipids to the parenchyma through the space of se) and physical contact with the parenchyma. The rate of delivery of sterols by parenchyma from Kupffer cells to the parenchyma may be slower than by direct uptake of liposomes by the parenchyma; the chemical form of sterols may be altered by Kupffer cells prior to metastasis; There is cell-cell communication; and based on other pathways for lipid transfer between liver cells, the process of Kupffer-to-parenchymal transfer can be controlled, but SUV crevices are unlikely to appear .

The second contributing explanation for the difference in metabolic response to LUV vs. SUV is based primarily on the difference in the kinetics of cholesterol delivery to the liver. LUV is SU
Somewhat slower than V, it is cleared from plasma, thereby producing a relatively constant delivery of cholesterol populations to the liver from the time of injection until most of the injected material is removed. SUVs are cleared more rapidly, thereby delivering a large bolus of cholesterol population to the liver for several hours after each injection. This is followed by a sustained rise in plasma concentrations of cholesterol esters and atherogenic lipoproteins. Slow, steady delivery by LUV avoids disruption of hepatic cholesterol homeostasis, while more rapid uptake of SUV cholesterol overwhelms the liver's ability to maintain homeostasis, thereby triggering inhibition of hepatic LDL receptors. To do. Other methods of delivering artificial particles or their components to the liver at appropriate rates also include, for example, altering the structure or composition of the particles, including specific ligands for charge and cell-specific binding. Is available by.

The third contributing explanation is based on the striking quantitative differences in protein adsorption between the two types of vesicles (FIG. 28), in certain embodiments, as a result of their different surface bending. is there. Therefore, SUV, not LUV, is apo from VLDL
Strip E, thereby showing its clearance from plasma and supporting conversion to LDL. SUVs that acquire apoE compete with VLDL, LDL and other particles for receptors that are mediated by hepatic uptake. Also, adsorbed apoproteins may play a role in directing phospholipid vesicles to different hepatic metabolic pools. Other methods of reducing proteins taken up by artificial particles are also available, for example by altering the structure or composition of the particles, including specific ligands for charge and cell specific binding.

Thus, assuming the observation that cholesterol ester and LDL concentrations do not increase after delivery of most of cholesterol and other exchangeable substances to the liver by LUV, the plasma concentration of atherogenic lipoproteins is What was a route of delivery to a specific metabolic pool or a pool with unique properties that did not increase or adversely impair hepatic cholesterol homeostasis, including regulation of genes and other functions, was: It was clear. Thus, these inventions are based on original particle components (eg, phospholipids), and substances acquired by the particles (
For example, cholesterol) is considered in part as a unique delivery system that brings specific delivery sites to harmless disposal and other additional benefits. Delivery systems with these characteristics are useful in any setting (regulation of hepatic cholesterol homeostasis, regulation of hepatic phospholipid homeostasis, and hepatic metabolism), and hepatic metabolism is generally advantageous .

For example, in situations where it is desired to modify red blood cell lipids, a straightforward approach is to administer artificial particles that can contribute and be removed from the appropriate lipid. However, when SUVs are used for this purpose, they transport cholesterol and other substances to the liver, to the bad pool, and / or at a bad rate in a detrimental manner, and SUVs are responsible for atherogenic lipoproteins. It results in an increase in plasma concentrations, which is an unwanted side effect that prevents this approach.
In contrast, the use of large liposomes or other particles with similar properties results in the proper delivery of the native and acquired substance to the appropriate pool at the appropriate rate. As a result, the desired effect (modification of erythrocyte lipids) can be achieved without a deleterious increase in the plasma concentration of atherogenic lipoproteins.

As another example, it may be desirable to modify infectious agents (eg, bacteria, fungi, and viruses) using the compositions and methods described herein. Administration of large liposomes or other particles with similar properties contributes to removed and replaceable substances to and from these infectious agents, and then the administered particles are suitable. To the desired pool, so that the desired effect can be achieved without deleterious elevation of the plasma concentration of atherosclerotic lipoproteins. The modifications of the infectious agent can be understood for the purpose of the example to reduce their pathogenicity, invasiveness, infectivity, transmissibility, fertility, and / or resistance to the immune system.

As another example, a beneficial treatment may induce a decrease in plasma levels of atherosclerotic lipoprotein as an unwanted side effect. Administration of large liposomes or other particles with similar properties modifies this response via delivery of lipids and other substances to the appropriate hepatic metabolic pool. The data using the "Mix" animals provide a specific example of this effect (Figure 4).

There are several mechanisms that affect arterial uptake, accumulation, and retention of lipoproteins. Liposomes are derived from atherogenic lipoproteins by apo
It captures E, thereby reducing lipoprotein binding to arterial cells and also competing for arterial cell binding. Finally, changes in the size and / or composition of LDL affect its binding to the extracellular matrix and are subsequently associated with deleterious changes within the arterial wall, such as susceptibility to oxidative or enzymatic modifications. Affect.

The action or mode of manipulation of large acceptors (eg, large liposomes) can be assisted by small acceptors, and vice versa, and this can be endogenous (eg, HDL) and exogenous (eg, aposomes). Both protein-phospholipid complex) small acceptors are applied. Large receptors seem to penetrate poorly into the interstitial space and inefficiently approach the cell surface under certain circumstances. These effects interfere with the uptake and contribution of exchangeable substances from membranes, cells, tissues, organs, and extracellular regions and structures. Small acceptors penetrate the interstitial space well and are accessible to the cell surface. This allows effective uptake of exchangeable substances. However, small acceptors have a major disadvantage. They have a very limited ability to acquire and contribute matter (even if their initial rate of acquisition and contribution is rapid until their ability is saturated), and once Once acquired, they deliver the substance to the liver in a manner that destroys hepatic cholesterol homeostasis.

However, large and small acceptors together synergistically overcome each other's weaknesses through at least three mechanisms. First, the large acceptor acts as a sink (or supply) of exchangeable material. The small acceptors, on the other hand, act in a different direction as shuttles that draw substances from peripheral storage to the large acceptors. So, for example, a small acceptor
It penetrates into the tissue, acquires (and / or contributes) substances from the tissue, and their capacity becomes at least partially saturated. They leave the tissue and encounter large acceptors in plasma, where the small acceptors are stripped of tissue lipids. The small acceptor capacities are thereby restored, so that when they return to the tissue, they may acquire (and / or contribute) more material. This cycle lasts many times. Second, a large acceptor can reconstruct some small acceptors. For example, the big acceptor is HDL
Can contribute phospholipids to the tissue, increasing the ability of HDL to acquire tissue cholesterol and other substances. Third, as described elsewhere, the presence of large acceptors may block or reduce the deleterious disruption in hepatic cholesterol homeostasis caused by small acceptors.

Large liposomes generally avoid elevated plasma levels of atherogenic lipoproteins as well as LDL. This list includes apolipoprotein B (a
all lipoproteins including poB) (eg LDL, IDL, VLDL,
Lp (a), β-VLDL, and residual lipoprotein).

Immune cells are also targets of depletion using the methods and morphologies of manipulation disclosed herein. Administration of HMG-CoA reductase inhibitor, pravastatin, to heart transplant recipients reduces the cytotoxicity of natural killer cells in vitro, reduces the incidence of rejection with hemodynamic compromise, reduces coronary vascular disease, and plasma. It is understood that it reduced LDL levels (and increased HDL levels) and significantly enhanced 1-year survival. The effect of survival was dramatic; in the control group, 22% died in the first year, while only 6% in the pravastatin treated group died.

The immune effects of HMG-CoA reductase inhibitors have been reported in vitro. They reported that immune effects included regulation of DNA in circulating cells, inhibition of monocyte chemotaxis, regulation of natural killer cell cytotoxicity, and inhibition of antibody-dependent cell cytotoxicity. Modulation of such inhibitors results from changes in circulating lipids or other effects and use of the methods and modalities of manipulation disclosed herein.

HMG-CoA reductase catalyzes an early step in cholesterol biosynthesis and is important for the synthesis of molecules in addition to cholesterol. HMG-CoA
Addition of cholesterol to immune cells treated with reductase inhibitors does not restore function, but addition of mevalonate does. This suggests that cholesterol deficiency is not a direct cause of immune effects, but the use of liposomes or other acceptors to remove cholesterol from cells has led to cells trying to produce more cholesterol. It is therefore shown to increase the endogenous consumption of mevalonate. The methods and treatments described herein also interfere with the ability of immune cells or other cells to make up for the loss of cholesterol by acquiring more LDL or otherwise lipoproteins, and to reduce plasma cholesterol. It is done with concentration-based therapies (HMG-CoA reductase inhibitors, fibric acid, niacin, bile acid binders, LDL pheresis, etc.).

These processes include enhanced cholesterol removal and reduced cholesterol influx. Levels of HDL, an apparent natural mediator of cholesterol removal from peripheral cells, were increased in treatment groups of patients and LDL
The level has decreased. Administration of HMG-CoA reductase inhibitors in vivo usually results in very slight changes in reductase enzyme activity:
Cells simply allow more enzymes to overcome the presence of inhibitors. They also make more LDL receptors, especially in the liver, so LDL levels are reduced.

The present invention further provides an additive to PD (peritoneal dialysate) that reduces the accelerated atherosclerosis that occurs in renal failure. The invention further provides a vasospasm disorder,
For example, treatments for Raynaud's phenomenon and related syndromes, and Printzmetal's angina and related syndromes are provided. The present invention also relates to hypercoagulable states such as TTP, other platelet disorders, DIC (so-called anti-phospholipid antibody syndrome, protein C abnormal state, protein S abnormal state, V-Leiden factor, skin patch-like vasculitis, lipopoiesis). Provide treatments for lipodermatosclerosis and related or related syndromes.

Monocyte chemotaxis is an important early phenomenon in joint disorders: monocytes are cellular cells created against abnormal arterial lipid deposits and in response to the presence of these deposits. For the product, it is attracted, enters the vessel wall and is converted into macrophages. Thus, monocyte chemotaxis and / or chemotaxis of other inflammatory cells can be achieved using the methods disclosed herein. Both cellular and humoral immunity appear to be affected by reductase inhibition: Cardiac rejection achieved by hemodynamic compromise is often associated with humoral rejection (ie, endocardial myocardium). Occurs without significant lymphocytic inflammation in the biopsy specimen).

Pravastatin may interact with cyclosporin, which is an important immunosuppressant, which blocks the synthesis of interleukin 2 in stimulated T lymphocytes. Addition of interleukin 2 restored the cytotoxicity of natural killer cells and partially restored the antibody-dependent cytotoxicity (inhibited by lovastatin treatment in in vitro cell culture). Synergistic use between cyclosporine and pravastatin increases immunosuppression in recipients of heart transplants (while non-transplant patients treated with HMG-CoA reducer senhibitor for hypercholesterolemia have clinical immunosuppression). Is not described).

Therefore, the use of a safe cholesterol acceptor with other immunosuppressive agents such as cyclosporine and / or glucocorticoids, which may also suppress IL-2, is also contemplated by the present invention. It

It is also understood that the present invention utilizes derivatives of various compounds described herein.

Pathological preparations from patients with heart grafts with severe coronary vasculopathy have been reported to have high cholesterol content. Thus, pravastatin-induced early cholesterol lowering may in part serve to lower cholesterol uptake into the coronary arteries of the donor heart. The same effect is achieved with large liposomes or other cholesterol acceptors (rapidly and directly, alone or in combination with them).

Immunomodulators are important in many conditions, not just heart transplants. Other areas in which the above approaches may be used are transplantation of other organs, autoimmune diseases (the body's immune system mistakenly attacks the body's own tissues), some infections (the immune response is detrimental). ), And any other circumstances in which immunomodulation is beneficial.

With regard to infection, modification of the lipid content and composition of foreign foreign bodies (eg infectious agents) in the body (although maintaining normal hepatic cholesterol homeostasis) should also be mentioned.

Oxidized lipids alter tissue function and cause damage, including decreased EDRF and increased adhesion molecules, cytotoxicity, and macrophage chemotaxis.

There is an interaction between LUVs and small acceptors such as HDL, apoprotein phospholipid complex, and cyclodextrin. Liposomes
Providing extra phospholipids reassembles HDL into a better acceptor. And the small acceptor acts as a shuttle, which efficiently transports cholesterol from cells to liposomes. LUV does not elevate LDL concentration and does not suppress hepatic LDL receptor gene expression. Medical benefits for LUVs include restoration of EDRF secretion by endothelial cells. High cholesterol levels inhibit endothelial release of EDRF by oxidized derivatives of cholesterol rather than by cholesterol. Liposomes can then do the same because HDL itself restores EDRF release, presumably by removal of cholesterol or oxidized lipids (eg, HDL passes cholesterol and / or oxidized lipids to liposomes).

The present invention provides methods of manipulation and modalities for modifying cellular lipids (including oxidized lipids) without inducing elevated LDL levels or detrimentally interfering with liver homeostasis. To do. Therefore, probably cholesterol (eg HDL)
LUVs that act in concert with the small (or extrinsic) acceptors of
It draws oxidized lipids out of the peripheral tissues and carries them to the liver for excretion. Oxidized lipids have a wide range of deleterious biological effects. The effect of this is E
Examples include suppression of DRF release, introduction of cell adhesion molecules, cell injury, and chemotaxis of macrophages. Therefore, inflammatory cytokines, growth factors, lipolytic enzymes, proteolytic enzymes, and / or nuclear factor kappa B (NF-κB) are induced. Both of these are regulated by the liposomes described herein.

Oxidized lipids and their deleterious effects include decreased endothelial C-type ANF, increased endothelial PAI-1, and decreased tPA and decreased endothelial thrombomodulin. Liposomes potentiate or participate in this effect. These changes impair the body's ability to dissolve blood clots. The methods disclosed herein help alleviate these deleterious effects of oxidized lipids. HDL
Acts in part by transporting enzymes that inactivate biologically active oxidized lipids.

It is understood that oxidised LDL inhibits endothelial secretion of C-type natriuretic peptide (CNP). It is the lipid component of oxidized LDL that mediates this effect. Most importantly, HDL blocks the action of oxidized LDL, presumably by taking up oxidized lipids (eg oxidized cholesterol). High density lipoproteins (which alone have no effect on CNP release (
Co-incubation with HDL) significantly interfered with Ox-LDL-induced inhibition of CNP secretion by endothelial cells (EC). Oxysterols, including 7-ketocholesterol, were analyzed in Ox-LDL by analysis by thin layer chromatography upon Ox-in the co-incubation of these two lipoproteins.
It was shown to be transferred from LDL to HDL. These results show that Ox-LDL
Shows that in Ox-LDL suppresses CNP secretion from EC by 7-ketocholesterol or other transferable hydrophilic lipids, and that the inhibitory effect of Ox-LDL is reversed by HDL.

No matter which molecule picks up HDL, remodeling of HDL by liposomes and shuttling of oxidized lipids by HDL from tissue to liposomes (reciprocation) (
That is, the liposomes continuously strip HDL), which allows the liposomes or other acceptors described herein to perform better. Liposomes with small endogenous acceptors also work.

It is further understood that transposable lipids in oxidized low density lipoprotein stimulate plasminogen activator inhibitor-1 and inhibit human tissue plasminogen activator from endothelial cells. . As mentioned above, this is a lipid in oxidized LDL (eg the oxidized form of cholesterol) that multiplies the effect. It is understood that the oxidized low density lipoprotein reduced thrombomodulin transcription in proliferated human endothelial cells. It is understood that oxidized lipids play a role in atherosclerosis and that enzymes on HDL that inactivate oxidized lipids may contribute to the protective effect. Methods and compositions disclosed herein provide additional platforms for enzyme transport and action, eg, by eliminating end products of these enzymes, otherwise altering HDL. Is further intended to help this proposed mechanism. For example, both paraoxonase and oxidized lipids can be transferred onto liposomes, where inactivation of the subsequently oxidized material can occur.

Thus, the use of large liposomes, which first extract lipids, to remove generally harmful lipids (here, lipid-oxidized lipids) from peripheral tissues, either directly or via HDL. , Perhaps inactivating them and then delivering them or their degradation products to circulating liposomes. Direct methods for assessing oxidative and oxidative damage in vivo include: 8-epi PGF ₂ α assay for lipids; 8-oxo-2′deoxyguanosine for DNA; general tissue To assess antioxidant enzymes in, and vitamin E, vitamin C, urate and antioxidant levels of reduced glutathione / oxidized glutathione.

Described herein are methods and modalities related to providing the effect of reversing lipid transport (including transport of extracellular and any normally compatible substances) from cells, organs and tissues. Has been done. It covers not only cholesterol alone, but also sphingomyelin, oxidized lipids, lysophosphotidylcholines, proteins and phospholipids. Some effects of oxidants include increased calcification in arterial cells, as described above and below.

Three potential differences between the large and small liposomes to explain the different effects on LDL and apo B levels include: fenestral penetration (LUV <
<SUV); clearance rate (LUV <SUV, which results in sustained release of cholesterol to the liver, which may be less destructive); protein adsorption (LUV << SUV). It is understood that unilamellar vesicles offer advantages over multilamellar vesicles. This is because, for example, the internal bilayer of phospholipids should be somewhat shielded and rely on internal diffusion to obtain or provide flip-flop and compatible material from the outside of the particle. is there.

Non-esterified cholesterol increases tissue factor expression by macrophages. This is very important because it is a macrophage-derived tissue factor that makes the substance released in an unstable manner. It strongly stimulates the blood clot to destroy plaque, which in turn blocks the blood vessels that lead to a heart attack. The methods and modalities of the manipulations and compositions of the present invention affect tissue factor expression by alterations in non-esterified cholesterol and / or other related factors.

Slight adsorption of proteins by large liposomes can be attributed to L
Affects DL levels and / or atherosclerosis: 1) Acquisition of apoE from VLDL by small liposomes impairs removal of VLDL from the circulation, thereby converting it more efficiently into atherogenic LDL Ii) Adsorption of proteins on small liposomes directs these particles to a poorly metabolized pool in the liver. Polyacrylamide gel electrophoresis shows that liposomes (actually small liposomes) increase the size of LDL. Liposomes are used to modify the size, composition and structure of LDL, reducing its atherogenic potential.

Other properties of LDL can be altered by administration of liposomes. For example, liposomes reduce surface non-esterified cholesterol; reduce surface sphingomyelin; replace surface sphingolipids with POPC (slightly oxidized); antioxidant added to liposomes prior to administration To replace LDL. These changes substantially alter the arterial entry, retention, modification and atherogenicity of LDL.

Controlled side effects focus on hepatic cholesterol metabolism, hepatic expression of genes involved in cholesterol metabolism and plasma concentration of cholesterol-rich atherogenic lipoproteins (primarily LDL) including apolipoprotein B. To be For example, the reverse transport of sphingomyelin alters hepatic cholesterol metabolism (cellular sphingomyelin affects the intracellular distribution of cholesterol and therefore its regulatory effect; Although it is a precursor to ceramide, which mediates signal transduction, large liposomes appear to avoid any problems in this area. The same is true for the reverse transport of the oxidized form of cholesterol (even non-oxidized cholesterol in suppressive LDL receptor gene expression is more potent).
Cyclodextrins do not take phospholipids.

Liposomes include any exchangeable lipid, in fact any exchangeable amphipathic or hydrophobic substance, including lipids or proteins or those having these characteristics. To take. These include sphingomyelin, oxidized or modified lipids such as oxidized sterols and phospholipids. Typically, such liposomes are capable of taking nonesterified cholesterol and other exchangeable substances from other lipid bilayers (eg, cell membranes) and lipoproteins. Liposomes also take up proteins and provide phospholipids. As an example, liposomes reduce the SM: PC ratio in lipoproteins, which include atherogenic lipoproteins, thereby reducing their atherogenesis. During and after modification of cell membranes, lipoproteins, etc., liposomes are removed from plasma mainly by the liver. Throughout this application, we refer to this general process as "reverse lipid transport" in tissue,
It is understood that any exchangeable substance in the blood, extracellular milieu or liposomes may be involved. Specific examples of exchangeable substances include non-esterified cholesterol, oxidized forms of cholesterol, sphingomyelin and other hydrophobic or amphipathic substances.

These molecules accumulate in vascular dysfunction and have deleterious effects (eg,
It mediates cholesterol, oxidized cholesterol, sphingomyelin and other substances (eg lysophospholipids) or aging (eg sphingomyelin). For example, oxidized lipids, especially sterols, alter many peripheral tissue functions, which stimulate calcification by arterial cells in atherosclerosis, and endothelial plus released by endothelial cells. Stimulating the activity of minogen activator inhibitor 1; other oxidized lipid products include:
Lysophospholipids, which stimulate endothelial expression of adhesion molecules that attract macrophages to the lesion, are listed, and sphingomyelin accumulates in some cell culture models of aging and, along with cholesterol, may cause some cellular changes . Other changes, such as oxidation, may also mediate or accelerate aging. Many of these molecules are taken up in vitro by liposomes such as cholesterol, sphingomyelin, and possibly oxidized cholesterol, and many by HDL (cholesterol, oxidized cholesterol, oxidized lipids), which are It has been shown that other molecules are likely to be addressed as well. However, in terms of total mass, the majority of the required material is non-esterified cholesterol, and secondarily with proteins. Alternatively,
By acquiring non-esterified cholesterol, the liposomes can reduce the amount of oxidized cholesterol that develops. This is because there are few starting materials.

The effective duration described herein should not be construed to exclude, for example, very long course of treatment, years of duration. Repeated treatment periods separated by week, month or year should also not be excluded.

Side effects include overload of liver with cholesterol or other substances acquired by liposomes; continuous changes in liver function, eg LD
It is associated with inhibition of L receptors, stimulation of intrahepatic cholesterol esterification, stimulation of hepatic secretion of atheromatous lipoproteins including apolipoprotein B, and impaired uptake of atheromatous lipoproteins by the liver from plasma.

As used herein, the term “endogenous” indicates that HDL originates from the body and is not administered. However, HDL and related acceptors can be administered.

The data show another difference between large and small liposomes in vivo. Prior to injection, the liposomes used in this experiment are essentially electrically neutral, as indicated by their lack of rapid migration through the agarose gel when an electric field is provided. (This does not imply that charged liposomes or other particles cannot be used.) Small liposomes take up proteins and other substances and become electrically charged: If an electric field is provided, it will move rapidly through the agarose gel. We ran an agarose gel of plasma samples from three groups of rabbits. Small liposomes became more mobile in these gels. Large liposomes remain virtually immobile, which shows a low charge density, reflecting a low protein content.

There are two explanations for the difference between large and small liposomes: 1) Small liposomes penetrate through the opening in the liver endothelium, but large liposomes do not (and thus large liposomes). Go to Kupffer cells, and small liposomes go to liver parenchymal cells and cause problems);
2) Large liposomes are known to be cleared by the liver somewhat slower than small liposomes (for no known reason), so that they cannot easily overwhelm the liver. The data on charge density give some explanation (less protein, and therefore slower or altered liver uptake).

Delivery of cholesterol to the liver by LUV per mg of phospholipid
It is actually more effective than V. One difference is that LUV delivery is stable over a long period of time after injection, while SUV delivery peaks and falls.

Some of the compositions described herein include: egg phosphatidylcholine; not crystallizing at body temperature (eg, they contain at least one double bond) but resistant to oxidation. Synthetic phosphatidylcholines (eg, they do not have many double bonds (eg, 1-palmitoyl, 2-oleoylphosphatidylcholine (abbreviated as POPC)); other natural or synthetic phosphorus. Lipids alone or as a mixture; any of the foregoing supplemented with or replaced by hydrophobic or amphipathic substances that further allow liposomes or micellar structures. The extruder is indeed much larger than the large liposomes. In particular, it is not the only conceivable method for making LUVs. Other methods known in the art are available or can be applied to the preparation of large liposomes in general and LUVs in particular.The difference in the starting lipids for making liposomes often results in particles of the desired size, lamellae. , And other features for these applications are required to be modified in manufacturing details.Multi-layered or slight pauci-lamellar vesci.
The migration of cholesterol and other exchangeable molecules into and out of the inner bilayer of cle) is not instantaneous, resulting in a single unilame.
It is also understood that llar) or slight lamellar vesicles are the preferred morphology.

As used herein, a dose is in the form of large liposomes,
It contains 10 to 1600 mg of phospholipid per kg of body weight. Other acceptable rates and doses described herein include plasma LDL concentration responses, lipid mobilization, and biological responses (eg, endothelial function, organ perfusion and / or function, and coronary or Cerebral event).

If there is a change in membrane composition as well as function, membrane composition assays or tissue composition assays may be used. Compositional assays should include lipids, proteins and other components.

HDL can take oxidised substances, and HDL-related enzymes can inactivate oxidised substances. Transfer of oxidised substances to liposomes allows disposal by enzymes (eg paraoxonase) that inactivate oxidised substances and also migrate onto liposomes when the liposomes are cleared from circulation. To do.

The separation of time depends on the actual dose of substance, which affects hepatic cholesterol homeostasis, whether or not cholesterol-lowering agents are administered simultaneously. Thus, for small liposome doses of about 300 mg / kg body weight, slight disintegration occurs even after a single dose, and high dose single administration
Further collapse can occur. Exemplary separations of time include 1 day to 1 month, but the detailed schedule is determined by monitoring liver cholesterol metabolism and plasma levels of LDL and other atherogenic lipoproteins.

The major macrophages involved in liposome clearance are Kupffer cells in the liver and macrophages in bone marrow or spleen. The catabolism in the present invention is a so-called alternative pathway for initiating the conversion of cholesterol to bile acids (macrophages are known to have at least cholesterol catabolism) or sterols (enzymatically modified). Modified or unmodified) to other cells (eg, liver parenchymal cells that then process the molecule), which include cholesterol and / or phospholipid bile and classical bile acid synthesis pathways. Including but not limited to direct secretion to.

[0154]   The method also describes herein the control of the effects of cellular senescence.

The present invention provides in vitro and in vivo assays for oxidation, assays for the oxidative susceptibility of plasma components, and modified HLDs that inhibit oxidation (by binding to oxidation products and / or its paraoxonase or other. To assess the efficacy of liposomal therapy by performing assays of its ability to inhibit cholesterol (via its antioxidant component) and its ability to recruit HDL or plasma or serum or blood to cholesterol and other exchangeable substances. Including means.

Large liposomes can cause the recruitment of some substances that are also trapped between cells, which is the extracellular region. This extracellular substance causes the following problems: a) When it comes into contact with cells or platelets, they modify their function, b
) Simply take a place.

The rate of cholesterol mobilization can be determined empirically. It is understood that the movement of liposome clearance is different in different species (LUV in mice
t _1/2 is about 8 hours, but about 27 hours in rabbits and longer in humans). Thus, the calculated rates can vary from species to species. 300 mg S
Based on the data on UV administration to rabbits, the peak rate of clearing liposomal cholesterol from plasma is between 3 and 6 hours post injection. The liposomes then elevated plasma non-esterified cholesterol to just over 2 mmol / L; the total plasma volume was estimated to be 90 mL in a 3 kg rabbit, at which time total liposome cholesterol was 180 μmol. The t _1/2 for SUV in these rabbits is about 20 hours, approximately 10% is 3
Cleared in time; therefore the peak rate of removal of liposomal cholesterol was approximately 2 μmol / h / kg, which caused a subsequent increase in plasma cholesterol ester concentration. Of note in other time periods after injection was the low rate of liposomal cholesterol cleared from plasma. Note also that the liver is the dominant organ for clearance but not the only organ for clearance.

A single dose of 300 mg LUV / kg to 20-22 g mice is calculated to mobilize approximately 2400 nmoles of cholesterol in the first 24 hours after injection. In contrast to the data with SUVs in rabbits, the first 24 hours of cholesterol mobilization in mice injected with LUVs was very stable. This was calculated to be about 4.7 μmol / hr / kg for this first 24 hours, which was actually greater than the 2 μmol / hr / kg figure, which was the peak rate. . This is not a fair comparison. Because the clearance of LUV in mice is 3 times faster than in rabbits. If 4.7 is divided by 3, then 2
Smaller 1.6 μmoles / hour / kg are obtained, which are incomplete estimates.
Human speed can be empirically determined. However, it is clear that LUVs deliver their cholesterol at a stable rate, while SUVs push lipids into the liver simply and quickly.

At body temperature, the most desirable liposomes are fluids within the limits of the bilayer,
This is called the liquid crystal state. Liposomes in the gel state, which are less fluid, are less desirable.

It is understood that non-esterified cholesterol stimulates macrophages and expresses more tissue factor, a substance known to cause blood clots. This explains the presence of abundant tissue factor in rupture-prone plaques. When ruptured, this plaque-prone plaque exposes tissue factor to plasma, causing a clot that can occlude blood vessels, causing heart failure. This is another example of aberrant cell function, which can be reversed by liposome removal of cholesterol.

Some human conditions are characterized by a unique lipid composition of tissues, cells, membranes and / or extracellular regions. For example, in atherosclerosis, cholesterol (non-esterified, esterified, and oxidized forms) as well as other lipids accumulated intracellularly and in the arterial wall and other extracellular regions. . These lipids have potentially deleterious biological effects, for example, by altering cell function and by narrowing blood vessel lumens and impeding blood flow. Removal of lipids offers many substantial advantages. In addition, cells, membranes, tissues and extracellular structures, for example, increase the content and type of antioxidants, reduce the amount of oxidised substances, and the content of substances resistant to oxidization. By increasing the composition of the invention benefit from compositions and modifications that include increasing resistance to oxidative and oxidative damage. During aging, cells have been shown to accumulate sphingomyelin and cholesterol, which alters cell function. These functions can be restored in vitro by removal of these lipids and replacement with phospholipids from the liposomes. A major obstacle to making similar lipid alterations in vitro has been the nature of lipids metabolized from tissues, cells, extracellular regions, and membranes. Natural particles (eg, high density lipoproteins) and synthetic particles (eg, small liposomes) capable of metabolizing peripheral tissue lipids have considerable disadvantages: they are those that interfere with hepatic cholesterol homeostasis. Lipids to the liver, resulting in elevated plasma levels of harmful lipoproteins such as low density lipoprotein (LDL), the major atherogenic lipoprotein.

The invention described herein provides methods and compositions relating to the "reverse" transport of cholesterol and other substances and compounds from peripheral tissues to the liver while controlling plasma LDL levels. .

Agarose gel electrophoresis of plasma samples from the last set of rabbits injected with LUV, SUV, or saline (these agarose gels are separated by their charge, which is of a certain type). Particles and different types of particles are not the same). Freshly made SUVs migrate agarose very slowly, indicating that the newly made liposomes have a very small charge. After injection into animals or after co-incubation with plasma or lipoproteins, SUVs pick up proteins from lipoproteins (
pick up). These proteins give the SUV more charge,
Substantially facilitate their migration through the agarose gel. After exposure to plasma, SUVs migrate through these gels faster than LDL.

This gel showed a substantial difference between LUV and SUV. As expected, SUVs migrated before LDL in these gels. However, LUVs migrated almost exactly to where the newly prepared liposomes without protein migrate. This result indicates that LUVs, unlike SUVs, do not readily pick up proteins from circulating lipoproteins.

There is direct proof of this difference between liposomes. Human HDL, which has the majority of proteins picked up by liposomes, is incubated with either LUV or SUV, then the liposomes are reisolated and their protein to phospholipid ratio assayed. S per amount of liposome phospholipid
UV picks up about 40 times more protein than LUV. This difference appears to be due to differences in surface curvature: SUVs are smaller, resulting in their surfaces being more abruptly curved and therefore more distorted, resulting in easier protein binding. Can be inserted into.

There are two most likely metabolic effects of the difference in protein uptake between the two types of liposomes, and are: VLDL has two metabolic fate: can it be removed from plasma before it is completely converted to LDL by a lipolytic enzyme, or this LDL
Can be completely converted to circulating LDL. SUVs remove apoE from VLDL, thereby slowing its clearance from plasma and helping its conversion to LDL. In contrast, LUV leaves apoE in VLDL, resulting in no increase in plasma LDL concentration.

2. Absorbed apoproteins may play a role in directing liposomes to different liver metabolic pools and / or rates.

Here are several methods for assaying the effect on oxidation in vivo: Catella F, Reilly MP, Delanty N, La.
wson JA, Moran N, Meagher E, FitzGerald
GA, Physiologic formation of 8-epi
-PGF2 alpha in vivo is not affected
by cyclooxygenase inhibition. Adv Pro
stalandin Thromboxane Leukot Res. 23
: 233-236, 1995. These authors are the end products of lipid oxidation,
8-epi-PGF ₂ α is described. They propose that this molecule can be used as a measure of lipid oxidative flux in animals. This is the antioxidant level (
Like dietary influences), thiobarbituric acid-reactive substances (some sugars interfere with this assay), and short-lived oxidation intermediates (which do not show the total flux of the substance being oxidized). , Which is superior to other conventionally used in vivo measurements of oxidation. By removing oxidised lipids from the periphery, administration of LUVs results in less total oxidative flux in vivo, 8-epi-PGF ₂
α is a suitable method to measure this lesser total oxidative flux; Ca
det J, Ravanat JL, Buchko GW, Yeo HC, Am
es BN, Singlet xygen DNA damage: chro
matographic and mass spectrometric a
analysis of damage products. Methods E
nzymol. 234: 79-88, 1994. These describe the final product of DNA oxidation, 8-oxo-2'-deoxyguanosine. as mentioned above,
This molecule can be used as a measure of DNA oxidative flux in animals. L
Administration of UV reduces the in vivo DNA oxidative flux, which is due to this D
A suitable method for measuring NA oxidative flux; and Xia E, R
ao G, Van Remmen H, Heydari AR, Richard
son A, Activities of antioxidant enzy
mes in various tissue of male Fisher
r 344 rats are alternated by food restr
motion. J Nutr. 125 (2): 195-201, 1995. Antioxidant enzymes in tissues are measured to show their deoxidizing capacity. LUV helps this. Antioxidant levels (vitamin E, ascorbate, urate); oxidized and reduced glutathione; and many other measurements can be used to assess peripheral oxidative and oxidative damage. These and other measures are also combined with LUV administration to assess the efficacy of treatment.

Other particles that mimic those properties of large liposomes act similarly, metabolizing and exchanging peripheral lipids and other exchangeable substances while avoiding deleterious disruption in liver cholesterol homeostasis. Deliver the substance. For example, they are compositions and structures that are slowly taken up by the liver and / or liver and endothelial windows of compositions and structures that do not readily acquire certain endogenous proteins.
It contains two large emulsion particles in order to penetrate the trae). Such emulsions can be made with or without proteins and can be made from phospholipids and neutral lipids such as triglycerides or another neutral lipid.

The present invention also provides pharmaceutical compositions comprising or consisting essentially of sized liposomes and compositions in which the liposomes are slowly taken up by the liver.

The present invention also includes a method of reverse transporting cholesterol from peripheral tissues to the liver in vivo while controlling plasma LDL concentration, which method comprises converting phospholipids substantially free of sterols during the treatment period. It comprises the step of transdermally administering a therapeutically effective amount of the various large liposomes made up, whereby the liposomes pick up cholesterol during the treatment period. This method involves the optional step of enhancing the tissue penetration of cholesterol acceptors and increasing the extraction of tissue cholesterol and other exchangeable substances by co-administration of effective amounts of the compounds. This compound is
It is selected from the group consisting of small cholesterol acceptors and agents that increase small endogenous cholesterol acceptors. In a variant, co-administration of this compound is concomitant with parenteral administration of large liposomes. In another variation, co-administration of compounds is separated from the therapeutically effective amount of parenteral administration of various large liposomes for a time and at an effective time. Effective time is about 1
It ranges from minutes to about 2 weeks.

In another aspect, the invention includes an improved method of reducing the lipid content of arterial injury, which method comprises administering a therapeutically effective amount of an agent to a subject from peripheral tissues in vivo. Inducing reverse transport of cholesterol to the liver.
The agent is selected from the group consisting of large liposomes composed of sterols and phospholipids substantially free of small acceptors; the subject's plasma LDL concentration is periodically monitored to obtain an LDL concentration profile; A therapeutically effective amount of the agent is adjusted in response to the concentration profile; and the pharmaceutical agent is administered to the subject. The agent is selected from the group of compounds for lowering LDL concentration, small acceptor, and compositions that increase HDL concentration in response to an LDL concentration profile, which results in a reduction in lipid content of arterial injury.
Effectively treated and monitored over the treatment period. Arterial injury includes lipid-rich, rupture-prone type IV and type V arterial injury. Puncture rupture, thrombosis, and tissue infarction are greatly reduced.

In yet another aspect, the invention provides an improved method of assessing the efficiency of a treatment for reducing the lipid content of an arterial injury, which injury contacts plasma and its components. The method comprises inducing reverse transport of cholesterol from peripheral tissues to the liver in vivo by administering to the subject a therapeutically effective amount of the drug. The drug is selected from the group consisting of large liposomes composed of sterols and phospholipids substantially free of small acceptors; plasma components are periodically monitored by the assay. This assay is for plasma non-esterified cholesterol and phospholipids, fecal bile acids and cholesterol assays, bile bile acids and cholesterol assays, liver gene expression assays in liver biopsies, peripheral It is selected from the group consisting of assays of gene expression in blood leukocytes, which genes include genes involved in cholesterol metabolism, assays of plasma LDL concentration, and vascular imaging techniques. Vascular imaging technologies include cardiac catheters, magnetic resonance imaging, ultrasound,
It is selected from the group consisting of ultrafast CT, radionuclide assays (including stress-thallium scanning, where appropriate), any perfusion assay (eg PET scanning), and any functional assay such as ECHO.

The invention also includes a method of beneficially altering arterial function, platelet function and controlling plasma LDL levels and hepatic cholesterol homeostasis in vivo, which method comprises:
Administering parenterally a therapeutically effective amount of a variety of large liposomes composed of phospholipids that are substantially free of sterols for the duration of the treatment, with or without the administration of other agents. Include. Other agents optionally include small acceptors and agents that reduce LDL. Optionally, the method includes the step of making a measurement of arterial function. This measurement includes blood flow, oxygen delivery, measurement of endothelial-induced relaxation factor, measurement of intracellular calcium concentration in arterial cells, measurement of arterial cell proliferation, assay of arterial enzyme, assay in the presence of calcium channel blocker, It is selected from the group consisting of arterial uptake, accumulation and maintenance assays of lipid proteins, liposomal arterial accumulation assays, liposomal arterial maintenance assays, gene product assays, and arterial cell function assays. The measurement of endothelium-induced relaxation factor is selected from the group consisting of a functional determination of endothelium-dependent arterial relaxation, a chemical determination of the production of endothelial relaxation factor, and an assay for nitric oxide synthase.

Methods of controlling plasma LDL levels, arterial function, hepatic cholesterol homeostasis and in vivo platelet function while beneficially altering platelet function are also included. This method involves parenterally administering a therapeutically effective amount of a large number of large liposomes, ie liposomes administered with or without other agents, consisting of phospholipids substantially free of sterols during the treatment. The step of performing is included. This method optionally includes the step of measuring arterial function. The measurement is selected from the group consisting of measurement of endothelial-induced relaxation factor, measurement of intracellular calcium concentration in arterial cells, measurement of arterial cell proliferation, assay of arterial enzyme, and assay of gene product. The measurement of endothelial relaxant is selected from the group consisting of functional determination of endothelium-dependent arterial relaxation and chemical determination of endothelial relaxant production.

Also included are methods of degrading cholesterol using macrophages (eg, hepatic macrophages) in vivo and affecting plasma components or structural aspects of arteries, which method provides a subject with a therapeutically effective amount. Including the step of administering the liposomes, the liposomes are substantially cholesterol-free and of a size and composition such that the liposomes can be taken up by and degraded by macrophages. Cholesterol is recruited by liposomes, giving rise to liposomes that are taken up and degraded by macrophages. The method can also include the step of periodically monitoring plasma components using the assay. This assay includes assays for plasma unesterified cholesterol and phospholipids, plasma cholesterol ester transfer protein activity assay, fecal bile acid and cholesterol assay, liver gene expression in liver biopsy, genes in peripheral leukocytes. It is selected from the group consisting of expression assays (which genes include genes involved in cholesterol metabolism), plasma LDL concentration assays and vascular imaging techniques.

In yet another aspect, the invention includes a method of delivering a drug in vivo, a method of preventing the deleterious division of hepatic cholesterol homeostasis, including the step of drug trapping. This drug is a liposome with low cholesterol,
It is selected from the group consisting of cholesterol-free liposomes, emulsions, liposomes initially ingested by hepatic parenchymal cells, emulsions initially ingested by hepatic parenchymal cells. The drug is selected from the group consisting of drugs with proteins and drugs without proteins to obtain trapped drugs. The method also includes administering a therapeutically effective amount of drug trapped during the treatment period. This administering step comprises slowly infecting the trapped drug. In many respects, the administering step comprises administering small doses of the drug at approximately discrete times to prevent adverse division in hepatic cholesterol homeostasis, and using low doses of the drug. The inclusion and thereby disruption of hepatic cholesterol homeostasis is prevented.

Plasma LDL levels, hepatic cholesterol homeostasis, arterial enzymes, arterial function,
And methods of controlling platelet function and altering plasma plate hormone production are also included. This method involves parenterally administering a therapeutically effective amount of a large number of large liposomes composed of phospholipids substantially free of sterols during the treatment period. The effective amount is administered in a dosage, and the dosage is selected from single and repeated doses. The method comprises the steps of diagnosing the efficacy of administration by making a measurement of hormone production and adjusting the effective amount in response to this measurement. The measurement of hormone production is an assay selected from the group consisting of an assay for thromboxane, an assay for prostacyclin, an assay for prostaglandins, an assay for leukotrienes, and an assay for their derivatives.

In a still-aspect, the present invention provides methods of increasing plasma HDL levels while controlling plasma LDL levels, hepatic cholesterol homeostasis, and hepatic gene expression. The method comprises parenterally administering a therapeutically effective amount of a first drug. This first drug contains a large number of small liposomes to elicit HDL levels during the treatment. The method then includes the step of co-administering a second agent. This second drug comprises large liposomes composed of phospholipids that are substantially free of sterols during the treatment period. This effective amount is administered in a dosage selected from single and repeated doses. Co-administration acts such that the small liposomes stimulate deleterious changes in hepatic cholesterol homeostasis and prevent an increase in plasma LDL. On the other hand, the first drug consists essentially of small liposomes, and the second drug consists essentially of large liposomes. This method also
Diagnosing the efficacy of the administration by making plasma HDL and LDL levels measurements before, during and after the treatment period.

Also described herein are methods of modulating plasma LDL levels, hepatic cholesterol homeostasis in vivo while altering cell membrane composition and function. The method comprises the step of parenterally administering a therapeutically effective amount of a plurality of large liposomes comprised of phospholipids substantially free of sterols during the treatment period. This effective amount is administered in a dosage selected from single and repeated doses. The method comprises co-administering a small acceptor selected from the group consisting of a small acceptor of cholesterol, an acceptor of sphingomyelin, an acceptor of lysophosphatidylcholine and an acceptor of lipids. This method measures membrane fluidity and transmembrane ion flux (this ion is calcium ion,
Optionally selected from the group consisting of sodium and potassium ions), membrane fragility assays, and diagnosing the efficacy of administration by performing membrane function assays.

In a further embodiment, the present invention is for treating angina entering the liver of a subject consisting essentially of liposomes selected from the group of unilamellar vesicles, multilamellar vesicles, combinations thereof and derivatives thereof. Pharmaceutical composition of.
These particles are selected from the group of particles substantially free of cholesterol and particles free of cholesterol.

The non-liposome particles are selected from the group consisting of triglyceride phospholipid emulsions. The emulsions include emulsions that are not rapidly taken up by hepatic parenchymal cells, emulsions that are not taken up by parenchymal cells for extended periods of time, and triglyceride-phospholipid-protein emulsions.

Also included in the invention is a pharmaceutical composition for reducing the size of arterial lesions that enter the liver of a subject consisting essentially of the drug trapped within the drug. This drug is from the group consisting of liposomes low in cholesterol, liposomes free of cholesterol, emulsions, liposomes slowly ingested by hepatic parenchymal cells, and emulsions slowly ingested by hepatic parenchymal cells. To be selected. The drug is selected from the group consisting of drug with protein and drug without protein.

The present invention also provides a pharmaceutical composition for controlling plasma LDL levels, hepatic cholesterol homeostasis, and hepatic gene expression while increasing plasma HDL concentration, which composition increases HDL concentration. It includes a first drug, which comprises a plurality of small liposomes for triggering, and a second drug, which comprises a large liposome composed of phospholipids substantially free of sterols.

In yet another aspect, a method of controlling cholesterol metabolism in hepatic parenchymal cells in a subject in vivo through cell-cell communication from Kupffer cells to parenchymal cells is included. The method involves administering to the subject a liposome composition. The liposome composition is selected from the group consisting of large unilamellar vesicles and large multilamellar vesicles. The liposomes have an average diameter of about 100-180 nanometers. LDL levels in the subject do not increase. The method also includes diagnosing the efficacy of controlling cholesterol metabolism by assaying an indicator in the subject. The indicator is selected from the group consisting of subject plasma LDL levels, subject liver gene expression, sterol excretion controlling cholesterol metabolism in hepatic parenchymal cells in the subject, and sterol excretion in the subject's bile. And adjust the dose in response to this assay.

The present invention further provides modalities of manipulation of atherogenic lipoproteins, cellular and extracellular structures modified by the compositions described herein that result in a beneficial physiological effect.

Diagnosis or conditions generally associated with endothelial function or dysfunction can be treated using the methods disclosed herein. By way of example, some of these conditions or diseases include hypertension, epilepsy, hyperviscosity syndrome, preeclampsia, and inflammation. Inflammation includes autoimmune and non-autoimmune conditions.

The methods of rapidly and substantially treating angina disclosed herein provide for at least three synergistic beneficial effects of liposome treatment (eg, large "empty" phospholipid vehicle- "LEV" -wherein , "Empty" refers to any conventional term which indicates that the encapsulated drug is not essential). These three beneficial effects include (
These include 1) recovery of endothelial dysfunction, (2) recovery of increased platelet reactivity, and (3) reduction of whole blood viscosity. All three of these are relatively quick and substantial treatment effects. These effects are particularly important when there are occlusive, fibroblastic, lipid-poor arterial lesions that are unlikely to disappear rapidly or substantially with alterations in lipid transport. In the case of the heart, the in vivo method may refer to angina and its associated signs and symptoms (eg, generally shortened breathing,
Decreased exercise tolerance, heart wall movement abnormalities, arrhythmias, and heart function). In the case of the brain, the treatments disclosed herein help alleviate transient cerebral ischemic attacks and their associated signs and symptoms (eg, neurological dysfunction). In the case of the lower extremities, the treatments disclosed herein ameliorate lameness and its associated signs and symptoms. Developmental organ perfusion and function are also assisted.

The intravenous route of administration may also be premixed prior to the time of simultaneous co-administration,
Or co-administration with red blood cells and other blood products, either immediately before or immediately after the administration of blood products (up to a week). Therefore, the present invention also contemplates the use of liposomes with blood transfusion products.

The approaches described herein are directed to other therapeutic agents directed at the arterial wall (vasodilators, agents that interfere with cell adhesion molecules, anti-inflammatory agents, agents that modify cytokines, antioxidant treatments). Drugs and inhibitors of arterial wall enzymes (eg acetyl CoA:
(Including cholesterol acyltransferase, lipase, myeloperoxidase, lipoxygenase and phospholipase)). It is contemplated that the use of liposomes herein results in a synergistic effect with each of these compounds, the others described herein, and in part the therapeutic agents. This is because liposomes act by a mechanism different from the one used by these therapeutic agents.

Various cardiovascular agents are also used in the methods of the invention. Examples of these drugs include blood modifiers, anticoagulants, antiplatelet agents, thrombolytic agents, and adrenergic blockers.
er), an adrenergic stimulant, an α / β adrenergic blocker, an angiotensin converting enzyme (ACE) inhibitor (for example, Quinapril (Acc
upril®, Parke-Davis), ramipril (Altac)
e®, Hoechst), Captopril, Benazepril (Loten)
sin (registered trademark), Novartis, trandolapril (Mavik (registered trademark), Knoll), fosinopril (Monopril (registered trademark), Br)
istol-Myers), lisinopril (Prinivil®, M
erck), moexipril (Univasc®, Schwarz)
, Enalapril (Vasotec® tablets, Merck), enaprilat (Vasotec®, iv, Merck), lisinopril (Z
estril®, Zeneca), its active metabolites, and / or its derivatives.

ACE inhibitors with calcium channel blockers and ACE inhibitors with diuretics can also be used with the present invention.

Angiotensin II receptor antagonists, including selective AT-I subtype angiotensin II receptor antagonists, also find use with the present invention (eg, Candesartan cilexetil (Ataca).
nd (registered trademark), Astra), Irbesartan (Avapro (registered trademark), Bristol-Myers Squibb or Sanofi), L
osartan (Cozaa®, Merck), Valsarta
n (Diovan®, Novartis), angiotensin II receptor antagonist with diuretics, its active metabolites, and / or its derivatives).

[0194]   Group I-IV antiarrhythmic agents are also used in the present invention, Group I antiarrhythmic agents.
Examples include the following: Cardioquin^TM(The common name is
It is a nisin), Ethmozine^TM, Mexitil^TM, Norspace^TM (Generic name is disopyramide), Procanbid^TM(General name is Proca
Inamide), Quiniaglute^TM, Quinindex^TM, Ryth
mol^TM, Tambocor^TM, And Tonocard^TM. Group II antiarrhythmic drugs
Examples include the following: Betaspace^TM(General name is Sotaro
), Brevibloc^TM(Generic name is esmolol), In
general^TM(Common name is propranolol), and Special^TM (The common name is Acebutrol). Examples of the group III antiarrhythmic drug are as follows.
The following are included: Betaspace^TM, Cordarone^TM, Covert ^TM And Pacerone^TM. Examples of group IV antiarrhythmic drugs include the following:
Is: Calan^TM(Common name is verapamil) and Cardizem ^TM (The common name is diltiazem). Various anti-arrhythmias used in the present invention
As vascular drugs, Adenocard (generic name is adenosine), Lano
xicaps^TM(Common name is digoxin), and Lanoxin^TM(one
The common name is digoxin).

Antilipidemic agent used in the present invention
nt) includes bile acid isolates, Fibric Acid derivatives, HMG-CoA reductase inhibitors, and nicotinic acid.

[0196]   Other drugs used in the present invention include β-adrenergic blockers,
Β-adrenergic blockers with urine, calcium channel blockers, diuretics, (
For example, metyrosine (carbonic anhydrase inhibitor, combinati
on) diuretics, loop diuretics, potassium-sparing diuretics, and thiazides
And related diuretics), hypertensive emergency drugs, muscle contractors, various cardiovascular drugs (eg methyl
Rosin (Demser^TM, Merck), Mecamylamine (Inversine ^TM , Merck), Fentolamine (Regitine^TM, Novarti
s), Abciximab (Reopro^TM, Centocor & Lill
y) Rauwolfia derivatives and combinations), vasodilators, coronary vasodilators and
Include peripheral vasodilators and combinations, and vasoconstrictors.

The invention described herein also provides a method of treatment of vascular dysfunction. Vascular dysfunction includes resistant vascular dysfunction, intramyocardial small artery dysfunction, conductance vascular dysfunction, and epicardial coronary dysfunction,
It is not limited to these.

Equivalents to the various angina discussed herein (ie, the effects of transient myocardial ischemia in addition to standard angina (eg, substernal discomfort; lethargy, pressure, Tightening, smothering, or choking
)) Includes by way of example: impaired mechanical, biochemical, and electrical function of the myocardium, failure of normal muscle relaxation and contraction, transient left ventricular failure (this standard sign). Is shortness of breathing, or decreased exercise tolerance), papillary muscle failure (including mitral regurgitation, ventricular contraction defocus, causing, for example, segmental dilation or dyskinesia) and / or Or a greatly reduced efficiency of myocardial pump function, a wide range of abnormalities in cardiomyocyte metabolism, function and structure, inability to oxidize fatty acids, conversion of glucose to lactate, intracellular pH and high energy phosphate (adenosine triphosphate). Resulting in reduced myocardial storage of phosphate (ATP) and creatine phosphate), defective cell membrane function, (potassium) Umbilical leak and sodium uptake by myocytes), characteristic electrocardiographic changes (eg, repolarization anomalies, as evidenced by T-wave inversion and later by ST-part replacement), electrical instability (This can cause ventricular tachycardia or ventricular fibrillation), sudden death as a result of ischemia-induced malignant ventricular arrhythmia, other areas (eg left shoulder, arms, forearm and hand ulnar surface, back) , Neck, jaw, teeth, and / or epigastrium).

The present invention also has beneficial effects in treating conditions that affect vascular anatomy. Vascular anatomical structures include, by way of example: vascular vessels,
Posterior capillary venules, venules, arterioles, capillaries, cavernous
sineous), arteries, veins, any vasculature that normally lines the endothelium, and / or any vasculature that normally lines the endothelium but is denutrified.

The invention disclosed herein is also understood to include treatment of a wide range of cholesterol metabolism, lipid metabolism, and / or aging related conditions. Further exemplary conditions include cancer and predisposition to cancer (eg, colon cancer (which also
(Can be prevented with cholesterol synthesis inhibitor aspirin or related compounds)
, And breast cancer); alopecia (including male pattern baldness); skin wrinkles (including prevention and reversal of wrinkles); gray hair (including premature graying); chemical or radiotoxicity (eg, , Derived from the removal of lipophilic toxins such as oxidative products); and conditions associated with the signs or symptoms of accelerated aging. Treatment is applied to human and non-human animals, including pets.

The treatments, compositions, and methods disclosed herein are also useful in treating Tangier disease and related conditions. Tangier disease, a genetic disease in which patients have abnormally low levels of plasma high density lipoprotein cholesterol with enlarged and yellowed tonsils and spleen, encodes the protein ATP-cassette binding protein 1 (ABC1) It has recently been discovered that it is associated with a deficiency in the enclade gene. ABC1 binds intracellular cholesterol and transports it to the cell membrane receptor responsible for transporting cholesterol to extracellular acceptors. Cells with ABC1 deficiency are unable to release cholesterol, even in the presence of excess extracellular acceptor. In contrast, cells with excess levels of ABC1 release a lot of cholesterol to extracellular acceptors, thus killing cells with cholesterol deficiency.

ABC1 is the first intracellular cholesterol transport protein identified to play a role in the first step of the reverse cholesterol transport process. Identification of this protein has created the opportunity for the identification of drugs that stimulate this process and enhance intracellular transfer of cholesterol to the cell membrane. At this point, the rate at which cells can eventually release cholesterol depends on the presence of sufficient extracellular acceptor molecules. The present invention is also useful in combination with drugs that enhance intracellular transfer of cholesterol to the cell membrane.

HDL, and in particular HDL1, is known to act as an acceptor of cellular cholesterol. However, HDL has a limited capacity to acquire cholesterol and acts as a more efficient acceptor of cholesterol in the presence of other molecules that can act as cholesterol reservoirs or sinks. A particularly efficient and cooperative way to enhance cellular cholesterol efflux is by the combination of HDL and empty phospholipid liposomes as described herein. In this context, small HDL molecules shuttle cholesterol from the cell membrane and transport cholesterol to liposomes. Liposomes are quite large and have a larger capacity for cholesterol. HDL then freely binds more cellular cholesterol and continues the shuttle function. In this situation, limited amounts of HDL lipoproteins may facilitate the removal of even more cellular cholesterol.

When the deficiency in ABC1 limits the cell's ability to transport cholesterol to the cell membrane, extracellular acceptor capacity is by no means a rate-limiting step in the cholesterol efflux process. However, in the presence of pharmaceutical agents that stimulate intracellular transport of cholesterol by AbC1 or other intracellular cholesterol transport proteins, the ability of HDL to accept all translocated cholesterol may be a limiting factor in the reverse cholesterol transport process. .

Accordingly, the present invention provides that the vesicles are hepatically fenestral.
Empty phospholipid vesicles with a sufficiently large mean diameter to exclude passage through (which is an aperture of about 100 nm), combined with a drug that stimulates ABC1 function or a similar intracellular cholesterol transport protein, Requires intravenous administration. Of course, other liposome compositions described herein can also be used. As a result of the combination, the extracellular cholesterol acceptor particles have an enhanced capacity for cellular cholesterol. The size of the phospholipid liposome acceptor particles prevents the uptake of shed cholesterol by the liver parenchyma. This avoids cholesterol from interfering with hepatic cholesterol homeostasis.
Instead, these particles are cleared by Kupffer cells, where cholesterol is finally removed in bile.

The present invention is particularly useful in treating patients suffering from any number of genetic conditions that produce very low levels of HDL cholesterol. Many of these conditions are
Makes patients susceptible to accelerated atherosclerosis and heart disease.

Although only a few preferred embodiments of the invention have been described herein above, it is understood that the embodiments may be modified and altered without departing from the central spirit and scope of the invention, Those skilled in the art will understand. Accordingly, the preferred embodiments described above herein are to be considered in all respects as illustrative and not restrictive,
The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all variations that come within the meaning and range of equivalency of the claims are to be embraced herein. Is intended.

[Brief description of drawings]

[Figure 1]   FIG. 1 is a side cross-section of lipoproteins and liposomes.

FIG. 2 shows liver mRNA levels (pg / pg for CETP, HMG-CoAR, LDL receptor, and 7a hydroxylase; and LDL ChE.
) Table is illustrated.

FIG. 3 illustrates plasma LDL cholesterol ester concentration in response to LUV, SUV or saline injection over time in one modification.

FIG. 4 illustrates plasma LDL cholesterol ester concentration in response to LUV, SUV or saline injection over time in one modification.

FIG. 5 illustrates LDL receptor mRNA levels in the liver in response to LUV, SUV or saline injections over time.

FIG. 6. H in liver in response to injection of LUV, SUV or saline.
3 illustrates MG-CoA reductase mRNA levels.

FIG. 7 illustrates cholesterol transesterification protein mRNA levels in the liver in response to LUV, SUV or saline injections.

FIG. 8: 7 in liver in response to injection of LUV, SUV or saline.
-Exemplifies alpha hydroxylase mRNA levels.

[Figure 9]   FIG. 9 illustrates important points regarding LUV and arteriosclerosis.

FIG. 10 illustrates plasma LDL non-esterified cholesterol levels in response to LUV, SUV or saline injections over time.

FIG. 11 illustrates plasma LDL esterified cholesterol concentrations in response to LUV, SUV or saline injections over time.

FIG. 12 illustrates LDL esterified cholesterol levels in response to LUV, SUV or saline injections.

FIG. 13: Plasma VLDL in response to LUV, SUV or saline injections.
The esterified cholesterol concentration is exemplified.

FIG. 14 illustrates plasma HDL esterified cholesterol levels in response to LUV, SUV or saline injections.

FIG. 15 illustrates plasma HDL esterified cholesterol levels in response to LUV, SUV or saline injections.

FIG. 16 shows LUVs in control mice or apoE knockout mice.
3 illustrates the time course of cholesterol transfer after injection.

FIG. 17 shows L in control mice or apoE knockout mice.
The time course of UV clearance is illustrated.

FIG. 18 illustrates that the compositions and methods of the present invention are effective in humans.

FIG. 19 shows a perspective view of the improved hemodialysis system of the present invention and an improved method of hemodialysis.

FIG. 20 illustrates a perspective view of an improved peritoneal dialysis system 2000 and a perspective view of a method of peritoneal dialysis.

FIG. 21 shows a modified 2100 modified peritoneal dialysis system with assay means.
And illustrates the method of peritoneal dialysis and analysis of the liquid used.

FIG. 22 illustrates an improved cardiac catheterization and / or angioplasty system 22.
00 is a perspective view and a method of cardiac catheterization and / or angioplasty.

FIG. 23 illustrates a perspective view of a modified cardiac catheterization and / or angioplasty system modification 2300 and a method of cardiac catheterization and / or angioplasty.

FIG. 24 illustrates a graph of liver fat content in response to LUV, SUV or saline injections.

FIG. 25 illustrates plasma free cholesterol concentration after repeated injections of SUV or LUV (300 mg / kg) into NZW rabbits.

FIG. 26 illustrates plasma cholesterol ester concentration after repeated injections of SUV or LUV (300 mg / kg) into NZW rabbits.

FIG. 27   FIG. 27 illustrates the changes in plasma components after repeated injections of SUV.

FIG. 28 illustrates an agarose gel electropherogram of whole plasma after repeated injections of LUV, SUV or saline.

[Procedure amendment]

[Submission date] May 20, 2002 (2002.5.20)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Contents of correction]

Title: Method for treating angina and / or angina equivalents, and pharmaceutical compositions and kits related thereto

[Claims]

Detailed Description of the Invention

Patent application to related parties The following is known. The present inventors, Dennis I., who is a US citizen. Goldb
erg (Destination 109 Bent Road, Sudbury, Massachus
setts 01776) and Kevin Jon Willi, a US citizen.
ams (Residence 425 Wister Road, Wynnewood, Penn
Sylvania 19096) invented a new and useful: “angina and / or angina equivalent state (equiva).
Lent) Method of Treatment, and Pharmaceutical Compositions and Kits Associated Therewith "The following is a detailed description of the invention of that specification.

[0002]   (A method of treatment of angina and / or angina equivalents,
And related pharmaceutical compositions and kits)   (Background of the Invention)   Chest pain can have many causes (eg, stomach discomfort, lung distress, pulmonary embolism, musculoskeletal pain). , Pneumothorax, non-coronary cardiac conditions, and ischemic coronary syndromes). Ischemic coronary symptoms
The groups include: stable angina, unstable angina, variant angina (angina
), Angina equivalence, myocardial infarction, and related dysfunction (eg, arrhythmia,
Low cardiac output, heart failure, infarct dilation, infarct extension, reperfusion injury, and coronary thrombosis).   Stable angina is angina that recurs in a regular and characteristic pattern. Human
Will only be able to play after some episodes and patterns
Recognize that you have Ginna. A certain level of activity that stimulates angina or
There is stress, and patterns generally change slowly.

Unstable angina is angina that manifests as either a very heavy episode or a frequently recurring angina attack. In unstable angina, the established pattern of angina can change sharply. That is, the angina may appear at rest or may result from far less exercise than in the past.

Atypical angina also refers to Prinzmetal's a angina.
ngina). This differs from the typical angina in that this angina occurs almost exclusively, when the person is at rest. Attacks can be very painful and usually occur between midnight and 8 am. Atypical angina, as well as other forms of angina and angina equivalents, can be associated with acute myocardial infarction, severe cardiac arrhythmias (including ventricular tachycardia, ventricular fibrillation , and even sudden death). Variant angina results from coronary artery spasm. This spasm can occur near atherosclerotic disorders. Many people with angina experience acute periods of activity.
Angina and cardiac events can often occur for 6 months or more.

Angina is defined as chest pain or discomfort of cardiac origin. This usually results from a transient imbalance between myocardial oxygen supply and myocardial oxygen demand. This discomfort is often induced by exercise, emotions, eating, or cold weather. Pain appears to be more likely to occur when the subject is outdoors (especially when the temperature is extremely hot or cold) and when the patient is walking uphill towards the wind . Angina generally occurs when a subject eats heavy metals or when the subject is exercising, angry, or nervous.

Normal coronary circulation is dominated and controlled by myocardial oxygen demand. This requirement is met by the ability of the heart to significantly fluctuate coronary resistance (and hence blood flow), while the myocardium extracts a high and relatively constant proportion of oxygen (Harrison's Principles of. Internal M
edicine, 12th edition, 1991, Chapter. 16). Normally, resistant arterioles in the myocardium show a vast capacity for dilation. During exercise and emotional stress, changes in oxygen demand affect coronary resistance and thus regulate blood and oxygen supply (metabolic regulation). These same blood vessels adapt to physiological fluctuations in blood pressure to maintain coronary blood flow at a level appropriate to myocardial demand (autoregulation). Although the epicardial coronary arteries can contract and relax, in healthy individuals they act as ducts and "conducting vessels" (cond).
uctance vesels). The intramyocardial arteries, on the other hand, usually show surprising changes in hearing and, therefore, "resistive vessels" (resis
(Tance vesels) (ibid.).

[0007] Once severe stenosis of the proximal epicardial artery reduces cross-sectional area by more than approximately 70%, distal resistant vessels dilate, reducing vascular resistance and coronary blood flow. To maintain. Pressure gradually develops across the proximal stenosis and post-stenotic pressure drops. When the resistant blood vessels are maximally dilated, myocardial blood flow becomes dependent on pressure in the coronary arteries far from the lesion. In these situations, changes in myocardial oxygenation may result from changes in myocardial oxygen demand and changes in the caliber of the stenotic coronary arteries due to physiological basal emotions, pathological convulsions, or small platelet plugs. All these transient events can reverse the important equilibrium between oxygen supply and oxygen reception, and thus lead to myocardial ischemia.

The effects of ischemia are many. Inadequate oxygenation induced by coronary atherosclerosis can result in transient impairment of myocardial medical, biochemical, and electrical function. Outbreaks of ischemia usually affect the segment of the left ventricular myocardium, with an almost instantaneous dysfunction of normal muscle relaxation and contraction. The relatively poor pericardial perfusion results in more intense ischemia of this part of the wall. Ischemia in a large segment of the ventricles reports transient left ventricular failure. And, when the papillary muscles are involved, mitral regurgitation can complicate this event. If the ischemic events are transient, they may be associated with angina; if prolonged, they may give rise to myocardial necrosis and scarring with or without the clinical symptoms of acute myocardial infarction. See: Harrison '
s Principles of Internal Medicine, 12t
h edition, 1991 Chapter. 189.

These underlying mechanistic disorders are a wide variety of abnormalities in cellular metabolism, function and structure. When oxygenated, normal myocardium metabolizes fatty acids and glucose to carbon dioxide and water. In severe hypoxia, fatty acids cannot be oxidized and glucose is broken down into lactate; intracellular pH decreases as myocardium accumulates high-energy phosphatases, adenosine triphosphate (ATP), and creatinine phosphate. To do. Impaired cell membrane function leads to potassium leakage and sodium uptake by muscle cells. The severity and duration of the imbalance between myocardial oxygen supply and myocardial oxygen demand determines whether the damage is reversible or whether it is homeostatic and subsequently leads to myocardial necrosis. Ha
rrison's Principles of lnternal Medic
ine, 12th edition, 1991.

Ischemia also causes characteristic electrocardiographic changes, such as repolarization abnormalities, as evidenced by T wave inversion and subsequent ST segment displacement (Harrison's Pris).
nciples of Internal Medicine, 12th ed
edition, 1991, Chapter. 176). Transient ST segment suppression often reflects subendocardial ischemia, while transient ST segment elevation is associated with a more severe transmural (tr).
It is considered to be caused by ischemia. Another important consequence of myocardial ischemia is electrical instability. Because it can cause ventricular tachycardia or ventricular fibrillation (Harrison's Principes of Internal).
Medicine, 12th edition, 1991, Chapter. 185
). Most patients who suddenly die from ischemic heart disease die of ischemia-induced malignant ventricular arrhythmias (Harrison's Principles of I).
internal Medicine, 12th edition, 1991, C
hap. 40).

The object of the present invention is to solve the problems associated with conventional therapies used to treat angina, angina equivalents and these various forms of acute coronary syndrome.

SUMMARY OF THE INVENTION The present invention provides a method of treating angina comprising the step of administering to a subject a therapeutically effective amount of liposomes. Stable angina, unstable angina, variant angina, and / or angina equivalent condition is treated by take advantage of the methods described herein. The liposome is selected from the group consisting of large liposomes, small liposomes and combinations thereof and is administered such that LDL levels in the subject are not substantially elevated.

When large liposomes are used, they are the chemical composition of the liposomes of size , function, composition or mode of administration or schedule such that the liposomes do not malignantly impair cholesterol homeostasis. Administering liposomes involves gradually injecting liposomes into a subject in one modification of the invention. In another modification of the invention, small amounts of liposomes are administered over time to avoid elevated LDL concentrations.

The method also includes periodically assaying plasma LDL concentration using an assay that obtains the assayed LDL concentration. This assay comprises a plasma esterified cholesterol assay, a plasma apolipoprotein B assay, a plasma gel filtration assay, a plasma ultracentrifugation assay, and components for determining the level of therapeutically effective amount of each of its liposomes. (Wherein its components are selected from the group consisting of polyanions, divalent cations, and antibodies), plasma ultracentrifugation assays, precipitation assays, immunoturbidimetric assays, and electrophoresis. Is selected from the group consisting of assays.

[0015] This method, a therapeutically effective amount of a liposome, used in a range of phospholipid / body weight kg / dose to about 10mg~ about 1600 mg. Liposomes are provided periodically during the treatment period in one embodiment. And it is selected from the group consisting of unilamellar liposomes, pauci-lamellar liposomes and multilamellar liposomes.

Preferably, the liposomes have a diameter greater than about 50 nm, a diameter greater than about 80 nm, and / or a diameter greater than about 100 nm. The liposomes also have a thickness of about 100 nm to about 150 nm, about 150 nm to about 200 nm.
From about 250 nm to about 300 nm, and / or from about 300 nm to about 40
It may have a diameter in the range of 0 nm. Other preferred liposome ranges are described herein.

In another embodiment of the invention, the liposomes are provided to the subject by intravenous bolus administration, intravenous injection, and / or intraperitoneal administration. Other routes are also contemplated (eg, intramuscularly, subcutaneously, intranasally, by inhalation, rectal and by oral or enteral absorption by encapsulation).

The method also includes modifications in which there is monitoring of the cardiac function of the subject. Typical cardiac functions monitored are EKG abnormalities, ST segment changes, arrhythmias,
Compartmental wall assessment, blood viscosity, exercise tolerance, ambulatory EKG monitoring, and / or heart wall motion abnormalities.

In yet another modification of the invention, the method comprises administering an effective amount of an anti-angina drug other than empty liposomes. “Empty” is a standard term indicating the absence of encapsulated drug within liposomes, where the encapsulated drug is essential for one or more functions of the liposome. is there. The anti- angina agents include nitrates, beta blockers, calcium channel antagonists, coronary vasorelaxants, lipid-lowering agents, post-load reducing agents, muscle contractors, pre-load reducing agents, and opiates.

As nitrates, for the purposes of illustration, nitroglycerin, sublingually administered nitroglycerin, long-acting nitrates, sublingual nitrate preparations, buccal (oral) nitrate preparations, oral nitrate preparations, spray nitrate preparations, oral nitrate spray, isosorbide dinitrate preparation, isosorbide-5-mononitrate preparations, sustained-release preparations of isosorbide-5-mononitrate, topical nitroglycerin, nitroglycerin ointment, Nitorogurise phosphorus-containing transdermal patch , And a nitroglycerin-impregnated silicon gel or polymer matrix.

As β-blockers, for the purposes of illustration, non-selective β-blockers, propanol, nadolol, pentabtrol, pindolol, sotalol, timolol, carteolol, drugs that block both β1 and β2 receptors, heart Mention may be made of selective β-blockers, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and drugs that block the β1 receptor with little effect on the β2 receptor.

In a modification of the invention, the calcium channel antagonist is a calcium antagonist, a compound that inhibits calcium ion migration through slow channels in the heart and smooth muscle membranes by non-competitive blockade of voltage-sensitive L-type calcium channels. , dihydropyridine, nifedipine ^TM, phenylalkyl amines, verapamil ^TM, benzothiazepine, diltiazem ^TM, nicardipine ^TM, amlodipine ^TM, and bepridil ^TM, calcium antagonists of the second generation, Nikarujiopin ^TM, isradipine ^TM, amlodipine ^TM, felodipine ^TM, and It is selected from the group consisting of dihydropyridine derivatives.

Yet another aspect of the invention is the step of administering an angiotensin converting enzyme (ACE) inhibitor to a subject in combination with a liposome, administering an antiarrhythmic drug to the subject in combination with the liposome, And / or performing a positive lifestyle in the subject. Typical positive lifestyle changes include weight loss, reduced smoking, loss of smoking, exercise, controlled exercise, decreased salt intake, decreased saturated fatty acid intake, decreased cholesterol intake, and total fat intake. Includes reducing, avoiding physical stress, avoiding emotional stress, and reducing caloric intake.

[0024] Yet a further aspect of the invention involves performing an anti-angina treatment in combination with liposome administration. Anti-angina therapy includes treatment of coexisting exacerbated conditions such as treatment for hypertension, treatment for hyperthyroidism, treatment for lung disease, treatment for heart failure, and treatment for anemia. ..

Another embodiment of the invention involves administering an antithrombotic therapeutic agent in combination with empty liposome administration. The antithrombotic treatment is selected from the group consisting of administering a therapeutically effective amount of an antiplatelet drug, administering a therapeutically effective amount of a drug that inhibits fibrin clot formation, and thrombolytic therapy.

Another embodiment of the invention includes a method of treating claudication. The method involves administering a therapeutically effective amount of liposomes.
As mentioned above, the liposomes are selected from the group consisting of large liposomes, small liposomes, and combinations thereof, and the method can be combined with the steps of the other methods described above and below.

Yet another aspect of the present invention includes a pharmaceutical kit for treating angina comprising: a first container 1 having liposomes and a second container having an anti-angina drug other than liposomes.

The present invention also relates to a method of acclimatizing a preoperative and / or intraoperative condition of a subject. This method involves administering liposomes alone or in combination with administration of anesthetics or sedatives, and / or preoperatively or intraoperatively assessing cardiac function in a subject.

Objects and features of the invention other than those specifically shown above will become apparent in the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION FIG. 1 illustrates a schematic diagram of the structure of normal lipoprotein 100 and unilamellar liposome 200. Lipoprotein 100 and liposome 200 include phospholipid molecule 300. Phospholipid molecules generally have a polar head 500 and a fatty acyl chain 400. Molecule 600 represents a non-esterified cholesterol molecule. Lipoprotein 100 comprises a hydrophobic core 102 composed primarily of triglycerides and cholesterol esters, surrounded by a monolayer of phospholipid molecules 300, whose fatty acyl side chains 400 face the hydrophobic core 102, The polar head 500 then faces the surrounding aqueous environment (not shown). Non-esterified cholesterol 600 is found primarily within phospholipid monolayers. Apolipoprotein 700 is located within phospholipid molecule 300. The artificial triglyceride emulsion particles have essentially the same structure, either with or without protein.

Liposomes 200 contain phospholipid molecules 300 to form phospholipid bilayers (eg, lamella, which may or may not contain proteins, fatty acyl side. The chains 400 face each other, their polar headgroups 500 on the outer leaflets are directed outwards towards the surrounding aqueous environment (not shown), and their polar headgroups 500 on the inner leaflets are aqueous on the particles 200. Inward towards core 202. Depending on the composition of particles 200, the phospholipid bilayer may have a large capacity for non-esterified cholesterol and other exchangeable materials and their components. There is no sterol, as illustrated in Figure 1. Typically, such liposomes are
Unesterified cholesterol can be picked up from other lipid bilayers such as cell membranes and from lipoproteins. Liposomes also pick up proteins and provide them to phospholipids and other exchangeable materials and their components.
Liposomes can also have multiple structures. Here, the bilayer is contained within an environment that is encapsulated by the outer bilayer to form multiple layers. The multiple layers may be arranged concentrically, like layers of onions, or in another modification, non-concentrically.

The liposomes described above are used in a method of treating angina and angina equivalents. Types of angina that can be treated using the method include stable angina, unstable angina, and / or atypical angina. In particular, the present invention is effective in treating angina.

The method includes the step of administering to a subject a therapeutically effective amount of a plurality of large liposomes, which comprises a phospholipid substantially free of sterols, to a subject. In one modification of the invention, the large liposomes are fenestratio of liver sinusoids lining the endothelial layer in the liver.
n) of larger size and shape. Preferably, the large liposomes have a diameter greater than about 50 nm, a diameter greater than about 80 nm, or about 100 n.
have a diameter greater than m. Particularly effective liposomes are from about 100 nm to about 20
It has a diameter in the range of 0 nm. In a modification of the invention, the liposomes are selected from the group consisting of large liposomes, small liposomes, and combinations thereof.

A therapeutically effective amount of liposomes is administered from about 10 mg to about 1600 mg phospholipids /
The body weight is in the kg / dose range, and the liposomes are given periodically during the treatment period. Therapeutically effective doses of liposomes may be given in a variety of ways, which illustratively include: intravenous bolus administration, intravenous infusion,
And intraperitoneal administration.

The method of administering liposomes can be combined with the step of determining efficacy of treatment. For example, the method involves monitoring the cardiac function of the subject before, during, or after administration of the liposomes. Pretreatment cardiac function measurements are performed prior to administration of the liposomes. After administration of the liposomes, an improvement in the patient's cardiac function may also be determined to indicate efficacy of the treatment and whether a subsequent treatment is needed, or the time or duration between subsequent treatments. The appropriate dosage for the treatment of can be determined.

There are many cardiac functions that can be analyzed in the present invention. These functions include EKG abnormalities, ST area changes, arrhythmias, evaluation of wall movement, blood viscosity, exercise tolerance, outpatient EKG monitoring, and heart wall movement abnormalities.

Liposomal administration can also be advantageously combined with administration of effective amounts of anti-angina agents other than liposomes, or anti-angina procedures such as LDL phoresis, hematopoiesis, or other revascularization. Anti-angina drugs include, for illustrative purposes, nitrates, beta blockers, calcium channel antagonists, coronary dilators, lipid lowering drugs, post-load reducing agents, muscle contractors, pre-load reducing agents, oxygen, opiates, and their Derivatives and / or combinations are included.It is understood that liposomal administration is also particularly desirable in patients (eg, diabetes) where conventional anti-angina therapies are poorly effective or ineffective.

Exemplary nitrates for use in the present invention include: nitroglycerin, sublingually administered nitroglycerin, long acting nitrates, sublingual nitrate preparations, buccal (oral) nitrate preparations, Oral nitrate preparation, spray nitrate preparation, oral nitrate spray, isosorbide dinitrate preparation, isosorbide-5-mononitrate preparation, isosorbide-5-mononitrate sustained release preparation, topical nitroglycerin, nitroglycerin ointment, nitro Included are glycerin-containing transdermal patches , and nitroglycerin impregnated silicone gels or polymer matrices.

Exemplary β-blockers used in the present invention include non-selective β-blockers, propanol, nadolol, pentabtrol, pindolol, sotalol,
Timolol, carteolol, a drug that blocks both β1 and β2 receptors, a cardiac selective β-blocker, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and β2 receptors with little effect on β2 receptors Drugs that can be used.

Exemplary calcium channel antagonists include compounds that inhibit the migration of calcium ions through slow channels in cardiac and smooth muscle membranes by non-competitive blockade of voltage-sensitive L-type calcium channels, dihydropyridine, nifedipine ^™ , phenyl. alkylamine, verapamil ^TM, benzothiazepine, diltiazem ^TM, nicardipine ^TM, amlodipine ^TM, and bepridil ^TM, calcium antagonists of the second generation, Nikarujiopin ^TM, isradipine ^TM, amlodipine ^TM, felodipine ^TM, dihydropyridine derivatives, their combination and their And derivatives thereof.

The invention also includes the step of administering an angiotensin converting enzyme (ACE) inhibitor to the subject and / or administering an antiarrhythmic drug to the subject to achieve a beneficial result for the subject. You can Various types of ACE inhibitors are described below. It is also possible to make positive lifestyle changes in the subject to obtain beneficial results. These lifestyle changes include weight loss, reduced smoking, loss of smoking, exercise, controlled exercise, decreased salt intake, decreased saturated fat and saturated fatty acid intake, decreased cholesterol intake, and total fat intake. Reductions, avoiding physical stress, avoiding emotional stress, and reducing caloric intake.

Yet another modification of the present invention, in combination with performing anti-angina therapy, is a therapeutically effective amount of a plurality of large liposomes comprising a phospholipid that is substantially free of sterol in the subject during the treatment period. (Eg empty liposomes). Exemplary anti-angina therapies include treatment of concomitant exacerbated conditions. For the purposes of illustration, coexisting exacerbated conditions include, for example, treatment for hypertension, treatment for hyperthyroidism, treatment for lung disease, treatment for heart failure, treatment for anemia, hypermetabolic conditions. And the treatment for diabetes.

Yet another aspect of the invention is to provide a therapeutically effective amount of a plurality of sterol-free phospholipids in the subject during the treatment period in combination with administering an antithrombotic therapeutic agent. Administering large liposomes (eg, empty liposomes). Various types of antithrombotic therapeutic agents are used in the present invention. This includes administering a therapeutically effective amount of an antiplatelet drug (described below), administering a therapeutically effective amount of a drug that inhibits fibrin clot formation, and / or thrombolytic therapy.

[0044] Large liposomes described herein (or, if deleterious effects of administration is administered in a manner that is substantially removed in modification, small liposomes described herein) Use of It is understood that it provides substantial LDL level regulation and / or regulation of other atherogenic lipoproteins. The methods described herein can be combined with monitoring LDL levels in a subject. When the liposome is a modification selected from the group consisting of large liposomes, small liposomes and / or combinations thereof, monitoring LDL levels in a subject, and more importantly, substantially increasing LDL levels in the subject. It may be desirable to administer the small liposomes in a manner that does not. This is achieved by injecting the liposomes slowly and / or by administering small doses of liposomes at separate times to avoid elevated lDL concentrations, or by administration of cholesterol-lowering agents or co-administration of large liposomes. obtain.

To monitor LDL concentration, plasma LDL concentration is regularly assayed using an assay that obtains the assayed LDL concentration. This assay
Plasma esterified cholesterol assay, plasma apolipoprotein B assay, plasma gel filtration assay, plasma ultracentrifugation assay, and component (eg, this component comprises a polyanion, a divalent cation, and an antibody. that obtained include sedimentation assay with selected). Other assays used herein include plasma ultracentrifugation assays, precipitation assays, immunoturbidimetric assays, and / or electrophoresis to determine the level of therapeutically effective amounts of each of the liposomes. An assay is mentioned.

It is also understood that the other method steps described above can be utilized with combinations of liposomes of different sizes , and that a particularly useful type of liposome comprises POPC. Liposomes containing phospholipids that are liquid crystals at body temperature but are still resistant to oxidation are desired. It is understood that liposomes transport cholesterol and other exchangeable materials and donate phospholipids, in part, in association with HDL and with transfer proteins such as phospholipid transfer proteins and cholesteryl ester transfer proteins. It These can be endogenous or exogenous.

In another aspect of the invention, pharmaceutical kits for treating angina are also described herein. The kit comprises a first container having liposomes; and a second container having an anti-angina drug other than liposomes. Exemplary anti-angina drugs are described above.

Yet another particular useful aspect of the invention involves a method of preoperative or intraoperative habituation of a subject, which method comprises administering liposomes. Many subjects are at risk of complications when undergoing surgery. Many of these risks can be reduced by administering liposomes at some effective, empirically determined time point prior to surgical procedures. Here, the patient's body is at risk of stress. The method of preoperative acclimation of the subject can be combined with treatments such that the risk of patient complications and / or stress associated with other known preoperative steps and surgical procedures can be minimized. These other pre-operative steps include, for illustrative purposes, administration of anesthetics or sedatives, pre-operative assessment of subject cardiac function, and the like.

The liposomes of the present invention are advantageously and efficiently manufactured using the novel methods and systems described herein. A desired substantially uniform size range (eg,
Generating liposomes of 125 nm to 250 nm) is particularly slow using conventional methods. This is because, during the initial fabrication, and because first single pass of the liposomes / liposome precursor is difficult, this is because large particles, in the conventional extrusion devices, passing slowly as glacier is there.

Accordingly, the present invention encompasses a commercial process for producing large quantities of liposomes within the desired size range. The method involves hydrating a phospholipid. This hydration step initially produces large particles that are difficult to pass through an extruder with a pore size in the desired range. Thus, this method utilizes high throughput equipment to bring the particles into a size range that readily passes through an extruder with a filter to produce the desired particle size (about 200 nm to about 400 nm). to conditioned pre hydrated liposomes, then followed by the pre-conditioning particles, passing the desired size (e.g., about 100nm size or other desired size) of extruder fitted with a filter Including steps. Exemplary high-throughput devices include microfluidizers, homogenizers, shear-based devices, and / or extruders with large pore sizes (500 nm or other suitable large pore sizes ). The above method, it is understood that it reduces or time of manufacture and / or processing in order to obtain liposomes of the desired range of sizes. The production rate bottleneck problem is eliminated by the preconditioning step described above.

There are populations of LDL that are considered to be particularly harmful (eg, the pattern βL
Low density LDL also known as DL). Pattern βLDL is commonly found in diabetes, hypertriglyceridemia, low HDL status, hypertension, insulin resistance, hyperinsulinemia and / or Syndrome X status. Low density LDL is characterized by poor lipids, especially with respect to surface lipids, including non-esterified cholesterol and phospholipids.
LDL can acquire phospholipids from liposomes. Treatment of the subject with phospholipid liposomes enriched for low density LDL and restored it to normal phospholipid content,
Reduce its atherogenicity and / or normalize its carbohydrate content and / or its size . Treatment with the compositions described herein includes
It alters the surface charge density of low density LDL and brings it to normal surface charge density. Lipoproteins in subjects with low density LDL show enhanced binding to proteoglycans (the strongest binding of that lipoprotein is IDL). The compositions and treatments described herein improve the deleterious properties of low density LDL and other lipoproteins commonly found in subjects with low density LDL.

Examples of deleterious properties ameliorated by the treatments disclosed herein include strong binding to the arterial matrix, easy oxidizability, ease of penetration into the arterial wall, and LDL receptors. Includes, but is not limited to, binding disorders.

The present invention further provides methods of treating the accelerated syndrome of vascular dysfunction.
Accelerated syndromes of vascular dysfunction include renal disease, transplanted atherosclerosis,
Asymptomatic ischemia, and its manifestations on all body parts (particularly brain, legs, genitals, and heart), ischemia, vascular grafts, diabetes mellitus, diabetes mellitus with nephropathy, diabetes mellitus with proteinuria Is included. The present invention also provides methods of treating other perfusion abnormalities such as hyperviscosity syndrome, sickle cell dyskeratosis, conditions involving impaired wound healing, and / or conditions involving aggregation of blood cells or platelets.

Organ perfusion can be determined herein by tests such as PET scans, Doppler evaluation, evaluation using contrast agents, and pulse wave examination.

Post-load reducing agents as used herein include antihypertensive agents and peripheral vasodilators (eg nitrate) and calcium channel antagonists.

The present invention also relates to male and / or female sexual dysfunction, acute coronary syndrome,
From stable coronary syndrome, intermittent claudication, transient ischemic attack, stroke, heart attack, myocardial infarction, myocardial ischemia, ischemia to any body part, organ failure from ischemia, and / or ischemia For treatment of organ infarction.

Administration of liposomes promotes angiogenesis, eg, formation of collateral vessels.
Lateral visual angiogenesis is a disorder in hypercholesterolemia and is affected by related particles containing LDL or apo-B. Therefore, the use of this method is a beneficial treatment for subjects who can benefit from accelerated angiogenesis, including collateral angiogenesis.

3 and 4 illustrate plasma LDL cholesterol ester concentration over time for LUV, SUV, or saline infusion. Rabbit with arrow 302
, 304, and 306, respectively, at days 1, 3, and 5 injected intravenously with a bolus of phosphatidylcholine 300 mg / kg body weight or a matching volume of saline. Its phosphatidylcholine, about 100 nm (preferably, about 120 nm) having a diameter of, large unilamellar particles (LUV) (LUV prepared by extrusion method, about the Nicomp ^TM model 370 submicron laser particle size determination apparatus 120 nm ( 123 ± 35 NM), and the extruded membrane had pores with a diameter of about 100 NM), or a small single particle with an ultrasonically prepared diameter of about 30 NM (preferably 35 NM). It was a pharmaceutical grade egg PC in either form of layered particles (SUV measured in the range 34 ± 30 NM). Blood was collected before each injection and at day 6 sacrifice. Plasma LDL cholesterol ester concentration was determined by plasma gel filtration assay by in-line enzyme assay for cholesterol ester. Mean ± SEM is shown in FIG. 3 animals injected with SUV
At days 5, and 6, there was significantly higher plasma concentration of LDL cholesterol ester compared to either LUV-injected or saline-injected animals. 2-8
, 10-15, 24, and 28 exemplify data from the same experiment in which infusions were made on days 1, 3, 5 and then the liver was removed. Gel filtration was performed on plasma to determine the lipid content of individual liposome classes. FIG. 2 shows C
ETP, HMG-CoAR (hydroxymethylglutaryl coenzyme A reductase), LDL receptor, and cholesterol 7-α-hydroxylase; and saline (HEPE for the experiments described with respect to FIGS. 3 and 4).
S-buffered saline) (rabbits 1-4), LUV (rabbits 5-8), and SU
Liver mRNA content for LDL ChE (low density lipoprotein cholesterol ester) for rabbits fed V (rabbits 10-12) (pg /
μg) table is illustrated. Rabbit 13 is a "Mix" rabbit.

FIG. 4 shows animals labeled Mix. "Mix" means the first day, the third day,
And a single animal that underwent SUV on day 5 and also received a single injection of LUV on day 3. Prior to this injection of LUV, the plasma concentration of LDL cholesterol ester was elevated, but after injection of LUV, the LDL concentration was decreased despite continuous administration of SUV.

FIG. 5 shows that, in response to injection of LUV, SUV, or saline over time,
3 illustrates LDL receptor mRNA levels in liver. The rabbits were sacrificed on day 6 and liver samples snap-stored in liquid nitrogen, mRNA was extracted and rabbit mRNA for the LDL receptor was quantified by an internal standard / RNase protection assay (Rea. T. J. et al., J
． Lipid Research 34: 1901-1910, 1993 and Pap. E. , Genet. Anal. 8: 206-312, 1991).
. Mean ± SEM is shown in FIG. Animals injected with SUVs showed significant suppression of liver LDL receptor mRNA compared to animals injected with LUVs or saline. Suppression of hepatic LDL receptor mRNA reflects the actual quality of being overloaded sterol and potentially harmful changes from normal hepatic cholesterol homeostasis. In contrast, LUV-injected animals show the highest levels of hepatic LDL receptor mRNA, but the increase over that observed in their saline-injected animals is not statistically significant. There wasn't. Livers from the above "Mix" animals showed a value of 5.28 pg LDL receptor mRNA / microgram. This is closer to the mean value in the saline group than in the SUV group. Therefore, LDL receptor mRNA was stimulated by a single injection of LUV despite repeated injections of SUV.

FIG. 6 illustrates HMG-CoA reductase mRNA levels in the liver in response to LUV, SUV, or saline injection. Details of this experiment are referenced above. SUV-injected animals had hepatic HMG-CoA reductase m compared to LUV-injected or saline-injected animals.
It showed significant suppression of RNA. Suppression of hepatic HMG-CoA reductase mRNA reflects the actual quality of being overloaded sterol, which can be a potentially harmful changes from normal hepatic cholesterol homeostasis. In contrast, LUV-injected animals showed the highest levels of hepatic HMG-CoA reductase mRNA, although an increase over that observed in saline-injected animals was statistically significant. It didn't reach.

“Mix” animals had 0.50 pg HMG-CoA reductase mRNA /
The microgram value is shown, which is the mean value (0.51) in the saline group.
, And is substantially higher than the value in the SUV group (0.27). Therefore, HMG-CoA reductase mRNA was stimulated to its normal value by a single injection of LUV, despite repeated injections of SUV.

FIG. 7 illustrates cholesterol ester transfer protein mRNA levels in the liver in response to LUV, SUV, or saline injection. The details of the experiment are referred to above. Animals infused with LUV compared to animals infused with SUV or saline with liver C
It showed significant suppression of ETP mRNA. Suppression of CETP mRNA resulted in alterations in plasma lipoprotein profile normally associated with reduced risk of atherosclerosis. "Mix" animals are 3.18 pg CETP
The value of mRNA / microgram was shown. This is closer to the mean value in the LUV group than in the SUV or saline groups. Therefore, CETP
mRNA was suppressed by a single injection of LUV despite repeated administration of SUV.

FIG. 8 illustrates cholesterol 7α hydroxylase mRNA levels in the liver in response to LUV, SUV, or saline injection. Details of the experiment are as described above. SUV-injected animals showed liver 7α-hydroxylase mRNA compared to LUV-injected or saline-injected animals.
Showed suppression of. Inhibition of 7α-hydroxylase can be a potentially deleterious change from normal liver homeostasis. In contrast, LUV-injected animals
Although it showed the highest levels of liver 7α-hydroxylase mRNA, the increase over that observed in saline infused animals was not statistically significant. “Mix” animals had 0.51 pg of 7α-hydroxylase mRNA
/ Microgram value was shown, which was higher than the mean value in the SUV group. Thus, 7α-hydroxylase mRNA was stimulated by a single LUV injection despite repeated SUV injections.

FIG. 10 shows non-esterified cholesterol levels in whole plasma over time in response to LUV, SUV, or saline. Details of the experiment,
As above. As shown in this figure, LUV and SUV significantly increased plasma concentrations of non-esterified cholesterol. This indicates immobilization of the tissue store. LUVs increased non-esterified cholesterol levels more than SUVs.

FIG. 11 shows esterified cholesterol concentrations in whole plasma over time in response to LUV, SUV, or saline injections . Details of the experiment are as described above. SUVs increased plasma levels of cholesterol ester on days 3, 5, and 6. FIG. 12 is a copy of the information contained in FIG.

FIG. 13 shows plasma VL in response to injection of LUV, SUV, or saline.
The DL esterified cholesterol concentration is shown. SUV is saline or LUV
Plasma concentrations of VLDL cholesterol ester were increased above those observed in the treatment group. "Mix" animals received 2.4 mg / dl plasma V on day 6.
The LDL cholesterol ester concentration was shown. This is lower than the average value in the SUV group. Details of the experiment are as described above.

[0068] Figures 14 and 15, LUV, SUV or the response to saline injection, shows the HDL esterified cholesterol concentrations. Details of the experiment are as shown in FIG. Suitable phospholipids are available from Avanti Polar Lipids, Nip.
pon Oil and Fat in Japan, and Princeto
n Lipids, as well as other vendors. LUVs are made by commercially available extruders. SUVs on Day 5 and 6
On day, it caused a small, but statistically significant increase in HDL cholesterol ester concentration.

FIG. 16 shows control or Jacjson Laboratories,
apoE KO (commercially available from In Bar Harbor, Maine)
Knockout) shows the time course of cholesterol immobilization after LUV injection in mice. Control (C57 / BL6) and apolipoprotein E knockout mice were infused with a single bolus of 300 mg LUV phospholipids / kg body weight at time zero. The LUV contained traces of labeled cholesterol hexadecyl ether. This remains on the liposomes after injection into mice. The data presented are for total cholesterol concentration in total plasma (ie esterified + non-esterified). The rise in both sets of animals is
We show that LUVs immobilize cholesterol in plasma even in the presence of severe genetic hyperlipidemia.

FIG. 17 illustrates the time course of LUV clearance in control and apoE mice. Details of this experiment are as described in FIG.
Clearance of plasma-derived LUVs was not diminished in apoE knockout mice, which indicates that cholesterol mobilization (FIG. 16) and placement (FIG. 17) even in the presence of severe genetic hyperlipidemia. ) Is shown. This shows the utility of this preparation in hyperlipidemia.

FIG. 18 illustrates another application for the compositions and methods of the invention in humans.

FIG. 19 illustrates a perspective view of the improved hemodialysis system and improved method of hemodialysis of the present invention. Among other benefits, this improved method allows for the treatment of angina, angina equivalent conditions, lameness, and related conditions, and allows pre-operative conditioning during or near the time of dialysis. . Blood was drawn from a site for circulatory access (shown here as arm 1900), and cells-
Transported to plasma separator 1910. This plasma is then transferred to the dialysis chamber 192
It is separated into at least two components that are transported to zero and separated by a semipermeable membrane 1930. One side of the membrane 1930 is patient plasma 1940 and the other side is dialysate 1950. The selected molecular exchange passes through the membrane 1930 depending on the characteristics of the membrane (charge, pore size, etc.). Device 1960 includes devices for adding a lipid acceptor to the dialysate and for sampling the dialysate, including cholesterol, phospholipids, and other components (eg, acceptors, specific lipoproteins, specific components). ) Assay and monitor placement. Extraction of plasma cholesterol or other extractables includes several possibilities: 1) the acceptor is located in the dialysate that does not pass from the membrane 1930 to the plasma; passes, and is separated from the or plasma returned to the patient remains present in the plasma, Ru der either then returned to the patient; and / or 3) a sheet (e.g., membrane 130 itself) Acceptors immobilized on top, on beads, and / or on the walls of chamber 1920. The plasma thus treated is usually returned to the patient after being remixed with blood cells. As shown, the cholesterol acceptor can be added at any stage as in the example, device 1970 includes the acceptor and simply adds the acceptor to plasma. Thereafter, returning plasma to the patient is also illustrated in FIG. Contaminating cellular substances in plasma (eg platelets)
It is further understood that cholesterol is depleted and phospholipids are enriched in endogenous lipids. All acceptors mentioned throughout this application can accept molecules in addition to cholesterol and donate substances as well.

The cell-plasma separator 1910-derived cell concentrate may then be treated in any of several ways and then returned to this patient: 1) returned to the patient without further treatment ( This involves mixing with plasma treated as above); 2) this dialysate containing cholesterol acceptors for lipids,
The cells are transported to a second dialysis chamber (not shown), which depletes the cells' endogenous lipids (eg, cholesterol), after which they are returned to the patient; 3) a suspension or solution of a lipid acceptor for the lipids. Mixed, depleting the cells of their endogenous lipids and then returning to the patient with the acceptor, or with further separation into specific cell types, or after this separation, option 1) and option 2) above. It can either be performed in all cell types or reverted (eg, purified platelets can be lipids depleted of endogenous lipids (eg, cholesterol) and enriched in liposomal lipids). . Options 2) and 3) are
Cellular cholesterol, phospholipids, flux, viscosity, fragility, cell composition and / or
Alternatively, periodic assays of cell function may be performed. Devices 1960, 1970
Includes devices that allow for periodic sampling of cells during treatment. Similar to plasma, lipid acceptors can be added at any stage of treatment. All fluids (
(Eg plasma and concentrated cells), specific gravity, mechanical, manually operated (syringe)
On or with a pump if necessary. Of course, it is understood that blood can be drawn for processing from any suitable part of the body.

FIG. 20 illustrates a perspective view of an improved peritoneal dialysis system 2000 and peritoneal dialysis method. Among other benefits, this improved method allows for the treatment of angina, angina equivalent conditions, lameness, and related conditions, and allows pre-operative conditioning during or near the time of dialysis. . Patient's abdomen 2010 (Fig. 20-
21) receives the peritoneal dialysate 2020 stored in the container 2030 into the peritoneal cavity via the channel 2050 and the incision 2040. The lipid acceptor and / or cholesterol acceptor 2060 may be added to the container 20 as needed.
70. In another refinement, the lipid acceptor is added to the dialysate 2020; simply added to the container 2030 in a concentrated form prior to infusion; added to the fluid stream entering the peritoneal cavity as shown; or Injected by a separate entrance to the patient by any effective route. It is understood throughout the application that all acceptors can accept molecules in addition to cholesterol and donate substances such as phospholipids and antioxidants.

FIG. 21 illustrates a perspective view of various improved peritoneal dialysis systems 2100 with assay means and methods of peritoneal dialysis and analysis of waste fluid. Vessel 2110 receives waste fluid from abdomen 2010 via channel 2120. Device 2110 is
It provides access to a diagnostic sample of waste dialysate and allows the assay of cholesterol, phospholipids, and other parameters described herein to demonstrate the efficacy of the described treatments. If desired, assay syringe 2130 is inserted into channel or tube 2120, or container 2110 via access inlet 2140. An optional pump (not shown) is then used to move the various fluids to the appropriate location in the assay.

FIG. 22 is a perspective view of an improved cardiac catheterization and / or angioplasty system 2200 and methods of cardiac catheterization and / or angioplasty in the context of angina or angina equivalent or associated ischemic injury. Is illustrated. Patient 2210 undergoes cardiac catheterization and / or angioplasty. This patient receives an effective dose of lipid or cholesterol acceptor 2230 intravenously co-administered with the treatment from container 2220. Intra-arterial access of catheters for coronary angiography and / or angioplasty allows preparation of co-administration of cholesterol acceptors and of diagnostic agents such as cholinergic agents to assess vascular function To

FIG. 23 shows a perspective view of various improved cardiac catheterization and / or angioplasty systems 2300 and cardiac catheterization and / or angioplasty in the context of angina, angina equivalent or other ischemic disorders. The figure is illustrated. The catheterization and / or angioplasty catheter 2310 has a device 2320 that allows the cholesterol acceptor to exit therefrom. In a modification, the catheter 2310 has a permeable membrane. This membrane allows the cholesterol acceptor to exit from it. Pseudo arrow 2330 indicates the exit site of the cholesterol acceptor and / or diagnostic agent. Site 2340 represents the acceptor or drug entry site. The balloons on device 2300 may be replaced or supplemented with other devices, or they may form an inner balloon layer located within an outer balloon layer. The acceptor is located between the inner flexible balloon layer and the outer flexible balloon layer. Upon inflation of the inner balloon layer, a force is exerted against the fluid or gel-like acceptor that forces the acceptor outside site 2320, and to a more localized direct treatment of arterial trauma ( Strongly)
Contact directly. It will be appreciated that various inventions provide maximum penetration of the acceptor at the site of the artery. This infusion can be accomplished by specific gravity, manual operation of the syringe, or by a mechanical infusion pump 2350. Similar methods and systems are
It can be utilized in standard vascular imaging techniques or in vessels including the femoral, carotid, and mesenteric vessels as an example.

Patient 2210 undergoes cardiac catheterization and / or angioplasty. The patient receives an effective dose of cholesterol or lipid acceptor 2230 intravenously co-administered with the treatment from container 2220. Intra-arterial access of catheters for coronary angiography and / or angioplasty provides for the preparation of co-administration of lipid or cholesterol acceptors and of cholinergic agents for assessing vascular function and / or organ perfusion. Allows preparation for administration of such diagnostic agents.

Container 2110 receives waste fluid from abdomen 2010 via channel 2120. Device 2110 provides access to a diagnostic sample of spent dialysate, which allows assaying of cholesterol, phospholipids, and other parameters described herein to demonstrate efficacy of the described treatments. If necessary, the assay syringe 2130 is inserted through the access portal 2140 into channel or tube 2120. An optional pump (not shown) is then used to move the various fluids to the appropriate location in the assay.

[0080] Figure 24 illustrates LUV, SUV or a graph of liver liquid content in the corresponding against the injection of saline. Details of the experiment are as outlined above.
Liver samples were assayed for the content of several lipids (cholesterol ester (CE); triglyceride (TG); non-esterified cholesterol (Chol); phosphatidylethanolamine (PE); and phosphatidylcholine (PC)) (these lipids. Is shown in units of μg (microgram) lipid / mg). Low values of PE and PC in SUV-treated animals were generated ; therefore, the Chol: phospholipid ratio in these animals was higher than in the other groups.

FIG. 25 illustrates cholesterol ester concentrations after repeated injections of SUV or LUV (300 mg / kg) in NZW rabbits (New Zealand white rabbits). Arrows indicate the time of phospholipid injection on days 0, 3 and 5. For a given phospholipid dose, LUVs promote a large increase in free cholesterol levels in plasma.

FIG. 26 shows SUV or L in NZW rabbits in the same experiment as FIG.
3 illustrates the free cholesterol concentration in plasma after repeated injections of UV (300 mg / kg). Arrows indicate the time of injection of phospholipids. Unlike SUV, L
Repeated injections of UV did not induce a dramatic increase in plasma CE concentration.

Elevated plasma CE concentration due to the delivery of excess cholesterol to the liver can be a continuous two process. This is due to overproduction of CE-enriched particles or CE
It may be involved in impaired clearance of enriched lipoproteins. The overproduction of CE-enriched particles that occurs after SUV injection can occur in plasma or in the liver. In plasma, LCAT acts for small or for a single layered phospholipid vesicles (with empty or encapsulated drug), or phospholipid enriched HDL generating CE, then, LDL by CETP Can be shipped over. Results using gel filtration of plasma from SUV-treated animals indicate that CE is transported mostly or substantially on LDL. In plasma, VL by SUV
Removal of apoE from DL slows the clearance of VLDL, thereby helping to more efficiently convert to LDL. In the liver, elevated delivery of cholesterol to hepatic cells during recruitment of cholesterol from surrounding tissues stimulates hypersecretion of apoB , CE-enriched lipoprotein.

[0084] In a variation, elevated plasma CE concentrations observed is the result of clearance scan was impaired CE enrichment atherogenesis Li <br/> lipoproteins. Intravenously administered liposomes, which acquire apoE, show LDL-receptor mediated uptake of L
Compete with DL. Delivery of excess cholesterol to specific regulatory pool in the liver, the LDL receptor was down-regulated, which interferes with hepatic uptake of atherogenic onset raw lipoproteins from plasma. For example, LDL removal from blood plasma is reduced; VLDL removed also impaired, and supporting lifting its conversion to IDL and LDL. The processes responsible for increasing plasma CE concentrations differ between the two liposomal preparations. Unlike SUV, LUV does not induce an increase in plasma CE concentration. LUV
Is an excellent preparation that mobilizes tissue cholesterol and other exchangeable substances but does not cause harmful side effects.

The methods and compositions of the present invention also provide for enrichment of HDL cholesterol ester by SUV. One contributing process is the stimulation of lecithin cholesterol acyltransferase (LCAT), and other processes associated therewith. The ability of SUVs to increase HDL cholesterol ester is LCAT
Is the result of stimulation and other processes associated with it. LCAT requires phospholipids and cholesterol to produce cholesterol esters and lysophosphatidylcholine; liposomes can supply excess phospholipids.
The invention also relates to changes in lipoprotein (LDL, HDL, etc.) composition,
And LUV and / or SUV and / or other acceptor modifications in function.

[0086] Method liposome compositions described herein and use of this liposome composition also takes the endogenous apoE, thus blocking the cellular up write only LDL, liposomes. Liposomes incorporate apolipoproteins, such as apoE and apoA-I, which alter or enhance their function. For example, uptake of endogenous apoA-I enhances the ability of liposome-derived phospholipids to uptake cholesterol. And the endogenous apo
Uptake of E allows liposomes to block specific pathways for arterial uptake of lipoproteins. All of these control <br/> control of LDL levels, and hepatic gene expression, and related to cholesterol homeostasis.

LUVs and SUVs deliver cholesterol to different regulatory pools within the liver. This conclusion indicates that differences in liver gene responses and CETP mRNA are suppressed; LDL receptor mRNA is unaffected or increased by LUV but suppressed by SUV; and CETP is suppressed by LUV. Supported by being unaffected by SUVs.

Important points regarding LUVs are illustrated in FIG. The practical advantage of using LUVs as a treatment for angina, angina equivalents and other ischemic disorders is that they are simple to manufacture, allergen free, synthetic and at very high doses. Even is non-toxic. Mechanically, LUV is LD
It promotes reverse cholesterol transport in vivo without inducing elevated L levels, and LUV is the optimal preparation. These particles are often referred to as "empty" vesicles, but drug uptake into the aqueous interior or bilayer of phospholipid vesicles also disrupts at least one essential action of the vesicle. It is understood that it can be implemented without.

[0089] The compositions used herein can be excluded are hepatic built actual quality or we direct clearance. And the various methods described herein, utilized with slow compositions injected described, thus, even if the clearance by the actual quality occurs, hepatocytes are not overloaded cholesterol . In addition, HDL is also CE
It is regulated by TP gene repression.

The assay is performed as described herein by: VLD
Assaying fasting plasma triglycerides to estimate L concentration; assaying plasma cholesterol (free ester, or total-free ester); precipitating LDL (and VLDL) with polyanion-cation; H
Assaying the supernatant, which is DL; and calculating the (total plasma value-VLDL-HDL) sterol (or sterol ester) of LDL in total plasma. Liposomes are precipitated with polyanion-cations; or, if desired, esters lacking most liposomes are assayed. Other assays include electrophoresis, chromatography, immunoassays, electron microscopy assays, functional assays, structural assays, and compositional assays.

In the dialysate of the invention, any liposome or emulsion can be used as long as they are cholesterol acceptors and they do not elevate LDL or they are returned to the circulation of the patient. In either case, the plasma LDL, and plasma concentrations of the acceptor, and is necessary to assay the plasma concentration of other atherogenic Li <br/> lipoproteins.

For methods requiring delivery of cholesterol to the liver at a slow rate or low dose, the administration is a small acceptor (eg, SUV) without LUV.
Although acceptable to use, but to monitor LDL levels as levels of other atherogenic lipoproteins and modulate. Drugs entrapped as described herein need not be given in low doses to avoid disrupting liver cholesterol homeostasis, and encapsulating liposomes or emulsions are given in low doses. The drug may be abundant in a few liposomes or a small amount of liposomal lipids.

Modifications in size, composition, and function of HDL can be achieved by administering high or even low doses of large and / or small liposomes with little or no sterol. Liposomes without sterols readily HDL and HDL when given at low doses.
It is split into apolipoproteins, and the pieces are then incorporated into the HDL fraction of plasma that enriches HDL in phospholipids. LUV for lower LDL levels
Or even drug-free SUV's such low doses (eg 10-
100 mg / kg / dose) is unlikely to increase plasma LDL levels,
Periodic monitoring is wise.

The method disclosed herein, which alters LDL composition without increasing LDL concentration, also uses a phospholipid such as POPC (palmitoyl oleoylphosphatidylcholine) that is resistant to oxidation. the composition enriched, enriched the composition with an antioxidant, deplete unesterified cholesterol, and reduce cellular or arterial uptake of oxidized LDL by phospholipid enrichment.

[0095]   Liposomes up to about 1000 NM are utilized in the present invention. Greater than Kina Liposomes can also be used, but make extraction of tissue lipoproteins inefficient.
Can be Further, the composition of the present invention can be concentrated or dried. Next
Then, these preparations are diluted or reconstituted at the time of treatment or administration.
In this variant, a two-component kit containing the active substance and a diluent is provided. Save
In order to avoid aggregation between the negatively charged liposomes or other
Encapsulation of phosphatidyl glycerol (PG) to create a charged component also
Provided.

FIG. 27 illustrates changes in plasma components after repeated injections of SUV. Watanabe hereditary hyperlipidemia (WHHL) rabbits receive 1000 mg / kg body weight of SUV phospholipids or an equivalent amount of saline intravenously over 3 weeks each Monday, Wednesday, and Friday (9 total doses). Gave to. Three days after the final dosing, blood samples were taken and plasma components were fractionated by size by passing through a Superose-6 gel filtration column. The eluate was read by an in-line spectrophotometer. The traces on the right are from saline injected rabbits and show approximately VLDL in fractions # 17-18, and approximately LDL in fraction # 27. The trace on the left is from a SUV-injected rabbit and shows approximately VLDL with persistent liposomes in fraction # 16, and approximately fraction # 25 LDL sized particles. This tracking is
Consistent with the increase in LDL, which is a detrimental effect, shows an increase in the amount of LDL sized particles following repeated injections of SUV. WHHL rabbits, because it has a genetic defect in the LDL receptor, this result, SUV, rather than by only suppressing the LDL receptor (Fig. 5), by an independent mechanism and LDL receptor (Figure 27) , indicating that destroy the liver warehouse cholesterol homeostasis. LUV avoids both LDL receptor-dependent and LDL receptor-independent destruction.

FIG. 28 shows agarose gel electrophoresis of whole plasma after repeated injections of LUV, SUV, or saline. Experimental details are cited by reference elsewhere in Figures 2-8 and elsewhere herein. 4 μL plasma samples from 2 rabbits in each group on day 6 were electrophoresed through 1% agarose and then stained with Sudan Black (O: origin, β: LDL standard migration). The SUV-mediated increase in LDL concentration is shown in these lanes by a darker, but otherwise unnoticeable β band. SUVs in plasma showed a mobility superior to LDL. This is mainly due to the acquisition of plasma proteins from HDL.
In contrast, plasma LUVs exhibit essentially the same mobility as freshly prepared protein-free vesicles (ie, just above the origin (O)), which is a substance parenchyma of the proteins acquired in LUVs. Absent or decreased in general.

Based on the electrophoretic mobility of FIG. 28, a quantification of protein acquisition by LUV versus SUV is obtained. LUVs and SUVs were incubated in vitro with human HDL at 37 ° C. for 4 hours, then separated from HDL by gel filtration chromatography and assayed for proteins and phospholipids. LUVs acquired 1.09 μg protein / mg liposomal phospholipids, while SUVs acquired 40.4 μg / mg (ie almost 40-fold). Therefore, the two types of liposomes show significant quantitative differences in protein adsorption. SUVs, but not LUVs, eagerly deprive VLDL of apoE, thereby slowing its clearance from plasma and favoring its conversion to LDL. Additionally, adsorbed proteins play a role in directing the SUV hepatic built metabolic pool to destroy the liver storehouse cholesterol homeostasis, whereas, LUV is not directed to such pool. Liposomes, emulsions, or any other particles or compounds that extract tissue lipids but do not acquire large amounts of plasma proteins behave like LUVs in these respects.

Specific vascular genes affected by cellular cholesterol loading include prolyl-4-hydroxylase; hnRNP-K; osteopontin (a role for oxidized lipids in inducing intra-arterial calcification). ); And the gene for Mac-2. The methods of modulating these genes described herein result in restoration of normal vascular or arterial function. Elevated expression of prolyl-4-hydroxylase (an enzyme in collagen synthesis, a component of fibrotic plaque) and hnRNP-K (identified in pre-mRNA metabolism and cell cycle progression) message is associated with aortic smooth muscle after cholesterol feeding. Found in cells. These normalize after liposome treatment as described herein. Other genes or enzymes that become abnormal with cholesterol loading are normalized by liposome treatment as described herein (osteopontin, nitric oxide synthase (NOS), adhesion molecules, chemoattractants, Tissue factor, PAI-1
(Plasminogen activator inhibitor), tPA (tissue plasminogen activator), and Mac-2 (Ramaley et al., 1995)).
Other genes affected by cholesterol, cholesterol load, oxidized lipids are also corrected.

Many examples of small acceptors, such as SUVs, apolipoprotein-phospholipid disks, and HDLs are commercially available and can be used in the present invention. Kilsdonk EP et al., Cellular cho
lesterol affiliated by cyclodex
trins, J. Biol. Chem. 270: 17250-17256, 19
95. As a further example, another small acceptor is cyclodextrin. Small acceptors (especially HDL) shuttle cholesterol from cells to liposomes. Cyclodextrin and also other small acceptors, the LUV from cultured cells, resulting shuttle cholesterol or other exchangeable material, which is substantially, given removal and preferred substances between cells and LUV (donation ) Increase. Apolipoprotein-phospholipid discs include, by way of example, wild-type apoprotein, apoprotein variants, apoprotein variants, apoprotein derivatives, and recombinant apoproteins.

Examples of antihyperlipidemic agents include fibric acid derivatives, HmG CoA reductase inhibitors, niacin, probucol, bile acid binders, other agents and combinations thereof. Antihyperlipidemic treatments also include LDL apheresis, ileal bypass, liver transplantation and gene therapy.

The data presented in this application support three possible tests for differences in metabolic response to LUV vs. SUV. The three mechanisms work either separately or in combination. To a 1, LUV, which are incorporated in the main by the Kupffer cells, SUV are mainly oriented in the liver parenchyma. This is a result of a partial mechanism of liver architecture: the liver endothelium window is an oval-shaped opening of approximately 100 × 115 nm, through which SUVs of 30 nm diameter can pass or easily. And access the real thing. Large particles (eg large liposomes of sufficient diameter) do not readily pass through and are instead cleared by macrophage Kupffer cells that line the sinusoids of the liver. SUVs are also accessible to Kupffer cells, but their sheer numbers (about 10 times the same SUV as LUV / mg of phospholipids ) appear to saturate the reticuloendothelial system, and Are dominant in their clearance . Other methods of orienting the artificial particles away from parenchymal also, for example, Tsu by to change the particle structure or composition (including the specific ligand for the charged us and cell-specific binding) Available.

[0103] In against the clearance pathway of cholesterol mediated by Kupffer cells, the cholesterol is substantially the result mediated clearance path has a different metabolism results. Direct delivery of cholesterol to the parenchyma by SUV suppresses sterol-responsive messages (FIGS. 5, 6 and 8). Delivery of cholesterol to Kupffer cells is followed by a gradual transfer of lipids to the parenchyma, eg, through Kupffer cell spreading (reaching through the Disse space), making physical contact with the parenchyma. Speed of delivery to real sterols transfer from Kupffer cells, slower it will obtain than by direct uptake of liposomes by substantially; chemical form of the sterol may be changed by the Kupffer cells before transfer; Other Based on other pathways for lipid transfer between liver cells, the process of Kupffer-to-parenchyma transfer can be controlled, but SUV clearance is likely to appear. Absent.

The second contributing explanation for the difference in metabolic response to LUV vs. SUV is based primarily on the difference in the kinetics of cholesterol delivery to the liver. LUVs are cleared from plasma somewhat slower than SUVs, thereby producing a relatively constant delivery of cholesterol populations to the liver from the time of injection until most of the injected substance is removed. SUVs are cleared more rapidly, thereby delivering a large bolus of cholesterol population to the liver for several hours after each injection. Thereafter, followed elevation lasted in plasma concentrations of cholesterol ester and atherogenic lipoproteins. Slow, steady delivery by LUV avoids destruction of hepatic cholesterol homeostasis, while more rapid uptake of SUV cholesterol overwhelms the liver's ability to maintain homeostasis, thereby triggering inhibition of hepatic LDL receptors. Other methods of delivering artificial particles or their components to the liver at appropriate rates also include, for example, altering the structure or composition of the particles, including specific ligands for charge and cell-specific binding. Is available by.

The third contributing explanation is based on the striking quantitative differences in protein adsorption between the two types of vesicles (FIG. 28), in certain embodiments, as a result of their different surface bending. is there. Therefore, SUV, not LUV, is apo from VLDL
Enthusiastically deprives E, thereby showing its clearance from plasma, and L
Support the conversion to DL. SUVs that acquire apoE compete with VLDL, LDL and other particles for receptors that are mediated by hepatic uptake.
Further, apoprotein adsorbed may play a role in Ru is directed phospholipid vesicles to different hepatic metabolic pools. Other methods of reducing proteins taken up by artificial particles are also available, for example by altering the structure or composition of the particles, including specific ligands for charge and cell specific binding.

[0106] In the cholesterol ester and LDL concentrations that do not increase after delivery of the majority of the cholesterol and other exchangeable material to the liver by LUV, assuming the observation, this delivery is atherogenic lipoprotein do not increase the plasma concentration, or (including control of genes and other functions) hepatic cholesterol homeostasis deleteriously infringement be that Germany it was delivery route to a specific metabolic pool with Japanese characteristics of the of clearly Met. Thus, these inventions allow the original particle components (eg, phospholipids) and substances acquired by the particles (eg, cholesterol) to be delivered to a particular delivery site for harmless disposal and other additional advantages. Partly regarded as the unique delivery system that results. Delivery systems with these characteristics are useful in any setting (regulation of liver cholesterol homeostasis, regulation of liver phospholipid homeostasis, and even in liver metabolism), and liver metabolism is generally advantageous.

For example, in situations where it is desired to modify red blood cell lipids, a straightforward approach is to administer artificial particles that can contribute and be removed from the appropriate lipid. However, if SUVs are used for this purpose, they will bring cholesterol and other substances into the liver, in a detrimental manner, into the bad pool, and / or
Or to feed transportation bad rate and SUV occurs an increase in the plasma concentration of atherogenic lipoprotein (a undesirable side effect of preventing this approach). In contrast, the use of large liposomes or other particles with similar properties results in the proper delivery of the native and acquired substance to the appropriate pool at the appropriate rate. that the desired effect without harmful increases in plasma concentrations of atherogenic lipoproteins (modification of erythrocyte lipids) can be achieved.

[0108] As another example, infectious factors using the compositions and methods described herein (e.g., bacteria, fungi, and viruses) may be desirable to modify the. Administration of large liposomes or other particles with similar properties, are removed, and the replaceable substances, these infectious agents, and to benefit from these infectious agents then were administered particles are suitably It is delivered to a pool, so that the desired effect can be achieved without harmful increases in plasma concentrations of atherogenic lipoproteins. The modifications of the infectious agent can be understood for the purpose of the example to reduce their pathogenicity, invasiveness, infectivity, transmissibility, fertility, and / or resistance to the immune system.

[0109] As another example, beneficial treatment can induce a decrease in plasma concentrations of atherogenic lipoproteins as undesirable side effects. Administration of large liposomes or other particles with similar properties modifies this response via delivery of lipids and other substances to the appropriate hepatic metabolic pool. The data using the "Mix" animals provide a specific example of this effect (Figure 4).

There are several mechanisms that affect arterial uptake, accumulation, and retention of lipoproteins. Liposomes, apo from atherogenic lipoprotein
It captures E, thereby reducing lipoprotein binding to arterial cells and also competing for arterial cell binding. Finally, the size of LDL and /
Alternatively, changes in composition affect its binding to the extracellular matrix and, subsequently, deleterious changes within the arterial wall, such as susceptibility to oxidative or enzymatic modifications.

The action or mode of manipulation of large acceptors (eg, large liposomes) can be assisted by small acceptors, and vice versa, and this can be endogenous (eg, HDL) and exogenous (eg, aposomes). Both protein-phospholipid complex) small acceptors are applied. Large accession Scepter is poorly penetrate the space stroma, and appear to inefficiently approach the cell surface under certain circumstances. These effects interfere with the uptake and contribution of exchangeable substances from membranes, cells, tissues, organs, and extracellular regions and structures. Small acceptors penetrate the interstitial space well and are accessible to the cell surface. This allows effective uptake of exchangeable substances. However, small acceptors have a major disadvantage. They, in order to acquire the material, or have very limited ability contribution to for (even those capabilities, until saturation, as the beginning of the speed of acquisition and contribution was rapidly), And, once the substances are acquired, they deliver them to the liver in a manner that destroys liver cholesterol homeostasis.

However, large and small acceptors together synergistically overcome each other's weaknesses through at least three mechanisms. First, the large acceptor acts as a sink (or supply) of exchangeable material. The small acceptors, on the other hand, act in a different direction as shuttles that draw substances from peripheral storage to the large acceptors. So, for example, a small acceptor
It penetrates into the tissue, acquires (and / or contributes) substances from the tissue, and their capacity becomes at least partially saturated. They leave the tissue and encounter large acceptors in plasma, where the small acceptors are stripped of tissue lipids. The small acceptor capacities are thereby restored, so that when they return to the tissue, they may acquire (and / or contribute) more material. This cycle lasts many times. Second, a large acceptor can reconstruct some small acceptors. For example, the big acceptor is HDL
Can contribute phospholipids to the tissue, increasing the ability of HDL to acquire tissue cholesterol and other substances. Third, as described elsewhere, the presence of large acceptors may block or reduce the deleterious disruption in liver cholesterol homeostasis caused by small acceptors.

[0113] Large liposomes, generally not only the LDL, avoid an increase in the plasma concentration of atherogenic lipoproteins. This list includes apolipoprotein B (a
all lipoproteins including poB) (eg LDL, IDL, VLDL,
Lp (a), β-VLDL, and residual lipoprotein).

Immune cells are also targets of depletion using the methods and morphologies of manipulation disclosed herein. Administration of HMG-CoA reductase inhibitors on cardiac graft recipients (pravastatin) decreased the cytotoxicity in vitro natural killer cells, reducing the incidence of rejection with hemodynamic compromise, reduced coronary vasculopathy reduces plasma LDL levels (and increased HDL levels), and the survival of one year it has significantly enhanced understood. The survival effect was dramatic;
In the control group, 22% died in the first year, while only 6% of the pravastatin treated groups died.

The immune effects of HMG-CoA reductase inhibitors have been reported in vitro. These control DNA to induce immune effects in circulation cells, inhibition of chemotaxis by monocytes, the regulation of cytotoxicity of natural killer cells, and reported that including inhibiting cytotoxicity of antibody-dependent cellular did. Modulation of such inhibitors results from changes in circulating lipids or other effects and use of the methods and modalities of manipulation disclosed herein.

HMG-CoA reductase catalyzes an early step in cholesterol biosynthesis and is important for the synthesis of molecules in addition to cholesterol. HMG-CoA
Adding cholesterol to the treated immune cells in reductase inhibitors include, but are not restore function, the addition of mevalonate is restored. This suggests that cholesterol deficiency is not a direct cause of immune effects, but the use of liposomes or other acceptors to remove cholesterol from cells causes cells to try to produce more cholesterol . It increases the endogenous consumption of mevalonate. More to interfere with the ability of immune cells or other cells to make up the loss of cholesterol by acquiring LDL or other lipoproteins, the methods and processes described herein also include low plasma cholesterol It is done with concentration-based therapies (HMG-CoA reductase inhibitors, fibric acid, niacin, bile acid binders, LDL pheresis, etc.).

These processes include enhanced cholesterol removal and reduced cholesterol influx. Levels of HDL, the apparent natural mediator of cholesterol removal from peripheral cells, were increased in treatment groups of patients , and LDL levels were decreased. Administration of HMG-CoA reductase inhibitors in vivo usually in reductase enzyme activity, occurs only very slight changes: the cells, a number of enzymes one surpasses the relative presence of the inhibitor than simply. They also make more LDL receptors, especially in the liver, so LDL levels are reduced.

The present invention further provides an additive to PD (peritoneal dialysate) that reduces the accelerated atherosclerosis that occurs in renal failure. The invention further provides a vasospasm disorder,
For example, treatments for Raynaud's phenomenon and related syndromes, and Printzmetal's angina and related syndromes are provided. The present invention also relates to hypercoagulable states such as TTP, other platelet disorders, DIC (so-called anti-phospholipid antibody syndrome, protein C abnormal state, protein S abnormal state, V-Leiden factor, skin patch-like vasculitis, lipopoiesis). Provide treatments for lipodermatosclerosis and related or related syndromes.

Monocyte chemotaxis is an important early phenomenon in joint disorders: monocytes are cellular cells created against abnormal arterial lipid deposits and in response to the presence of these deposits. For the product, it is attracted, enters the vessel wall and is converted into macrophages. Thus, inhibition of monocyte chemotaxis and / or chemotaxis of other inflammatory cells can be achieved using the methods disclosed herein. Both cellular and humoral immunity appear to be affected by reductase inhibition: Cardiac rejection achieved by hemodynamic compromise is often associated with humoral rejection (ie, endocardial myocardium). Occurs without significant lymphocytic inflammation in the biopsy specimen).

[0120]   Pravastatin smells in stimulated T lymphocytes
Cyclosporin that blocks the synthesis of interleukin 2 [important immunosuppression medicine It is possible to interact with. Interleukin 2AdditionIs a natural killer
Restores the cytotoxicity of cells to induce antibody-dependent cellular cytotoxicity (in vitro cell culture
, Which was inhibited by lovastatin treatment). Cyclos poly
Synergy between prawn and pravastatinProductImmunity in heart transplant recipients
Increased repression (while HMG-CoA reductase for hypercholesterolemia
Non-transplant patients receiving inhibitors do not have clinical immunosuppression)
To be done.

Therefore, the use of a safe cholesterol acceptor with other immunosuppressive agents such as cyclosporine and / or glucocorticoids, which may also suppress IL-2, is also contemplated by the present invention. It

It is also understood that the present invention utilizes derivatives of various compounds described herein.

Pathological preparations from patients with heart grafts with severe coronary vasculopathy have been reported to have high cholesterol content. Thus, pravastatin-induced early cholesterol lowering may in part serve to lower cholesterol uptake into the coronary arteries of the donor heart. The same effect is achieved with large liposomes or other cholesterol acceptors (rapidly and directly, alone or in combination with them).

Immunomodulators are important in many conditions, not just heart transplants. Other areas in which the above approaches may be used are transplantation of other organs, autoimmune diseases (the body's immune system mistakenly attacks the body's own tissues), some infections (the immune response is detrimental). ), And any other circumstances in which immunomodulation is beneficial.

[0125] For infection, foreign substances in the body (e.g., infectious agent) modifying the lipid content and composition (however, while maintaining normal hepatic cholesterol homeostasis) should also be mentioned.

Oxidized lipids alter tissue function and cause damage, including decreased EDRF and increased adhesion molecules, cytotoxicity, and macrophage chemotaxis.

There is an interaction between LUVs and small acceptors such as HDL, apoprotein phospholipid complex, and cyclodextrin. Liposomes
Providing extra phospholipids reassembles HDL into a better acceptor. And the small acceptor acts as a shuttle, which efficiently transports cholesterol from cells to liposomes. LUV does not elevate LDL concentration and does not suppress hepatic LDL receptor gene expression. Medical benefits for LUVs include restoration of EDRF secretion by endothelial cells. High cholesterol levels inhibit endothelial release of EDRF by oxidized derivatives of cholesterol rather than by cholesterol. Liposomes can then do the same because HDL itself restores EDRF release, presumably by removal of cholesterol or oxidized lipids (eg, HDL passes cholesterol and / or oxidized lipids to liposomes).

The present invention provides methods of manipulation and modalities for modifying cellular lipids (including oxidized lipids) without inducing elevated LDL levels or detrimentally interfering with liver homeostasis. To do. Therefore, probably cholesterol (eg HDL)
Is (also exogenous) endogenous LUV acting in concert with a small acceptor of,
It draws oxidized lipids out of the peripheral tissues and carries them to the liver for excretion. Oxidized lipids have a wide range of deleterious biological effects. The effect of this is E
Examples include suppression of DRF release, introduction of cell adhesion molecules, cell injury, and chemotaxis of macrophages. Therefore, inflammatory cytokines, growth factors, lipolytic enzymes, proteolytic enzymes, and / or nuclear factor kappa B (NF-κB) are induced. Both of these are regulated by the liposomes described herein.

Oxidized lipids and their deleterious effects include decreased endothelial C-type ANF, increased endothelial PAI-1, and decreased tPA and decreased endothelial thrombomodulin. Liposomes potentiate or participate in this effect. These changes impair the body's ability to dissolve blood clots. The methods disclosed herein help alleviate these deleterious effects of oxidized lipids. HDL
Acts in part by transporting enzymes that inactivate biologically active oxidized lipids.

It is understood that oxidised LDL inhibits endothelial secretion of C-type natriuretic peptide (CNP). It is the lipid component of oxidized LDL that mediates this effect. Most importantly, HDL blocks the action of oxidized LDL, presumably by taking up oxidized lipids (eg oxidized cholesterol). High density lipoproteins (which alone have no effect on CNP release (
Co-incubation with HDL) significantly interfered with Ox-LDL-induced inhibition of CNP secretion by endothelial cells (EC). Analysis by thin layer chromatography, oxysterols, including 7-keto-cholesterol, in Ox-LDL, during co-incubation of these two lipoproteins, ox-
It was shown to be transferred from LDL to HDL. These results show that Ox-LDL
Shown but Ri by the 7-keto-cholesterol, or other transferable hydrophilic lipids in Ox-LDL, it suppressed the CNP secretion from EC, and that the inhibitory effect of Ox-LDL is reversed by HDL .

No matter which molecule picks up HDL, remodeling of HDL by liposomes and shuttling of oxidized lipids by HDL from tissue to liposomes (reciprocation) (
That is, the liposomes continuously strip HDL), which allows the liposomes or other acceptors described herein to perform better. Liposomes with small endogenous acceptors also work.

It is further understood that transposable lipids in oxidized low density lipoprotein stimulate plasminogen activator inhibitor-1 and inhibit human tissue plasminogen activator from endothelial cells. . As mentioned above, this is a lipid in oxidized LDL (eg the oxidized form of cholesterol) that multiplies the effect. It is understood that the oxidized low density lipoprotein reduced thrombomodulin transcription in cultured human endothelial cells. It is understood that oxidized lipids play a role in atherosclerosis and that enzymes on HDL that inactivate oxidized lipids may contribute to the protective effect. Methods and compositions disclosed herein provide additional platforms for enzyme transport and action, eg, by eliminating end products of these enzymes, otherwise altering HDL. Is intended to help this proposed mechanism as well . For example, both paraoxonase and oxidized lipids can be transferred onto liposomes, where inactivation of the subsequently oxidized material can occur.

Thus, the use of large liposomes , which first extract lipids, to remove generally harmful lipids, here lipidated lipids, from peripheral tissues is directly
Alternatively, either via HDL, presumably inactivating them and then delivering them or their degradation products to circulating liposomes is described. Direct methods for assessing oxidation and oxidative damage in vivo include:
These include: 8-epi PGF ₂ α assay for lipids; 8-oxo-2′deoxyguanosine for DNA; antioxidant enzymes in tissues in general; and vitamin E, vitamin C. , Antioxidant levels such as urate and reduced glutathione / oxidized glutathione.

Described herein are methods and modalities related to providing the effect of reversing lipid transport (including transport of extracellular and any normally compatible substances) from cells, organs and tissues. Has been done. This does not include only cholesterol, sphingomyelin, oxidized lipids, Li zone phosphotidyl choline, proteins and phospholipids also provide cover. Some effects of oxidants include increased calcification in arterial cells, as described above and below.

[0135] As a difference with three possibilities in the large liposomes for small lipoic saw beam for describing the different effects on LDL and apo B levels include the following: opening penetration (fenestral penetration) (LUV
<<SUV; Clearance rate (LUV <SUV, which allows LUV to deliver sustained release of cholesterol to the liver (which may be less destructive)
Protein adsorption (LUV << SUV). It is understood that unilamellar vesicles offer advantages over multilamellar vesicles. Because, for example, the internal bilayer of phospholipids should be somewhat shielded and rely on internal diffusion to obtain or provide flip- flops and compatible substances from outside the particle. Is.

[0136] non-esterified cholesterol increases the tissue factor expression that by the macrophages. This is very important . Because it is a macrophage-derived tissue factor that makes the substance released in an unstable manner. It strongly stimulates the blood clot to destroy plaque, which in turn blocks the blood vessels that lead to a heart attack. The methods and modalities of the manipulations and compositions of the present invention affect tissue factor expression by alterations in non-esterified cholesterol and / or other related factors.

[0137] Slight adsorption of proteins by large liposomes, Ru <br/> by the following mechanism, acting on LDL levels and / or atherosclerosis: 1) Sumo acquisition of apoE from VLDL by Le liposomes impairs the removal of VLDL from the circulation, thereby making it possible to convert it more efficiently to atherogenesis LDL; ii) adsorption of proteins to small liposomes of these particles metabolism in the liver Oriented to a bad pool. Polyacrylamide gel electrophoresis shows that liposomes (actually small liposomes ) increase the size of LDL. The size of LDL using liposomes, change the composition and structure, reducing its atherogenic.

[0138] Other properties of LDL is could be changed by administration of liposomes. For example, liposomes reduce surface non-esterified cholesterol; reduce surface sphingomyelin; replace surface sphingolipids with POPC (slightly oxidized); antioxidant added to liposomes prior to administration To replace LDL. These changes, arterial entry of LDL, residence, substantially altering modification and atherogenic.

[0139] Side effects to be controlled, hepatic cholesterol metabolism, atherosclerosis onset accent lipoproteins rich in cholesterol including liver expression and apolipoprotein B of genes involved in cholesterol metabolism (mainly, LDL) focus on plasma concentration of Applied. For example, the reverse transport of sphingomyelin alters hepatic cholesterol metabolism (cellular sphingomyelin affects the intracellular distribution of cholesterol and therefore its regulatory effect; Although it is a precursor to ceramide, which mediates signal transduction, large liposomes appear to avoid any problems in this area. The same is true for the reverse transport of the oxidized form of cholesterol (even non-oxidized cholesterol in suppressive LDL receptor gene expression is more potent). Cyclodextrins do not incorporate phospholipids.

Liposomes include any exchangeable lipid, in fact any exchangeable amphipathic or hydrophobic substance, including lipids or proteins or those having these characteristics. Take in . This includes sphingomyelin,
Included are oxidized or modified lipids such as oxidized sterols and phospholipids. Typically, such liposomes will be associated with other lipid bilayers (eg, cell membranes).
And non-esterified cholesterol and other exchangeable substances from lipoproteins can be incorporated . Liposomes also take up proteins and provide phospholipids. As an example, liposomes, SM in lipoproteins: decreased the PC ratio, the this include atheroma onset accent lipoproteins, thereby reducing the atherogenic. During and after modification of cell membranes, lipoproteins, etc., liposomes are removed from plasma mainly by the liver. Throughout this application, we understand that this general process is "reverse lipid transport", but that any exchangeable substance in tissue, blood, extracellular milieu or liposomes may be involved. To be done. Specific examples of exchangeable substances include non-esterified cholesterol, oxidized forms of cholesterol, sphingomyelin and other hydrophobic or amphipathic substances.

These molecules accumulate in vascular dysfunction and have deleterious effects (eg,
It mediates cholesterol, oxidized cholesterol, sphingomyelin and other substances (eg lysophospholipids) or aging (eg sphingomyelin). For example, oxidized lipids, especially sterols, alter many peripheral tissue functions, which stimulate calcification by arterial cells in atherosclerosis, and endothelial plus released by endothelial cells. Stimulating the activity of minogen activator inhibitor 1; other oxidized lipid products include:
Lysophospholipids, which stimulate endothelial expression of adhesion molecules that attract macrophages to the lesion, are listed, and sphingomyelin accumulates in some cell culture models of aging and, along with cholesterol, may cause some cellular changes . Other changes, such as oxidation, may also mediate or accelerate aging. Many of these molecules, liposomes in vitro (e.g., cholesterol, sphingomyelin, and perhaps, oxidized cholesterol) is taken up by,
And although many are taken up by HDL (cholesterol, oxidized cholesterol, oxidized lipids), which was shown to be other molecules is to capture as well. However, in terms of total mass, the majority of the required material is non-esterified cholesterol, and secondarily with proteins. Alternatively, by acquiring cholesterol unesterified, liposomes, can reduce the amount of oxidized cholesterol occurs. This is because there are few starting materials.

The effective duration described herein should not be construed to exclude, for example, very long course of treatment, years of duration. Repeated treatment periods separated by week, month or year should also not be excluded.

Side effects include overloading of liver with cholesterol or other substances acquired by liposomes; subsequent changes in liver function, eg LD
Inhibition of L receptor, intrahepatic cholesterol esterification of stimulation, stimulation of hepatic secretion of Atero beam generating Li lipoproteins containing Aporipotanpa <br/> click protein B, and damage Atero beam generating Li lipoproteins by the liver from plasma Accompanied by uptake.

As used herein, the term “endogenous” indicates that HDL originates from the body and is not administered. However, HDL and related acceptors can be administered.

The data show another difference between large and small liposomes in vivo. Prior to injection, the liposomes used in this experiment are essentially electrically neutral, as indicated by their lack of rapid migration through the agarose gel when an electric field is provided. (This does not imply that charged liposomes or other particles cannot be used.) Small liposomes take up proteins and other substances, and become electrically charged: If an electric field is provided, it will move rapidly through the agarose gel. We ran an agarose gel of plasma samples from three groups of rabbits. Small liposomes became more mobile in these gels. Large liposomes, remains not substantially less mobile, indicating a low charge density which reflects the low protein content.

[0146] Two descriptions of differences between the large liposomes and small liposomes exist: 1) Small Liposomes penetrate through the openings of the liver endothelium, La chromatography di liposomes do not penetrate (therefore, large liposomes , it goes to the Kupffer cells, and small liposomes to go to the liver real quality, and cause problems)
2) Large liposomes are known to be cleared by the liver somewhat slower than small liposomes (for no known reason), so that they cannot easily overwhelm the liver. The data on the charge density give a partial explanation (less protein and hence slow or altered liver uptake).

Delivery of cholesterol to the liver by LUV per mg of phospholipid
It is actually more effective than V. One difference is that LUV delivery is stable over a long period of time after injection, while SUV delivery peaks and falls.

Some of the compositions described herein include: egg phosphatidylcholine; not crystallizing at body temperature (eg, they contain at least one double bond) but resistant to oxidation. Synthetic phosphatidylcholines (eg, they do not have many double bonds (eg, 1-palmitoyl, 2-oleoylphosphatidylcholine (abbreviated as POPC)); other natural or synthetic phosphorus. Lipids alone or as a mixture; any of the foregoing supplemented with or replaced by hydrophobic or amphipathic substances that further allow liposomes or micellar structures. The extruder is indeed much larger than the large liposomes. In particular, it is not the only conceivable method for making LUVs. Or other known methods are available, or generally the difference in the starting lipid for making large liposomes and may be particularly applicable for the production of LUV. Liposomes often desired sized particles, layered, and vesicles (Vescicl in order to obtain other features for these applications are understood to require a detailed modified manufacturing. multilamellar or a few layers (pauci-lamellar)
from inside the bilayer to or vesicles within the e) to the outer, rather than the movement of cholesterol and other exchangeable molecules moment, as a result, a single layer shape (Unilamella
It is also understood that r) or minor lamellar vesicles are the preferred morphology.

As used herein, a dose is in the form of large liposomes,
It contains 10 to 1600 mg of phospholipid per kg of body weight. Other acceptable rates and doses described herein include plasma LDL concentration responses, lipid mobilization, and biological responses (eg, endothelial function, organ perfusion and / or function, and coronary or Cerebral event).

If there is a change in membrane composition as well as function, membrane composition assays or tissue composition assays may be used. Compositional assays should include lipids, proteins and other components.

[0151] HDL is obtained uptake of oxidized substances and HDL-associated enzymes, it is an oxidized material may inactivate. Transfer of oxidised substances to liposomes allows disposal by enzymes (eg paraoxonase) that inactivate oxidised substances and also migrate onto liposomes when the liposomes are cleared from circulation. To do.

The separation of time depends on the actual dose of substance, which affects hepatic cholesterol homeostasis, whether or not cholesterol-lowering agents are administered simultaneously. Thus, for a dose of small liposomes of approximately 300mg per kg body weight, it caused even after a slight collapse single dose and high dose of a single dose may result in further disruption. Exemplary separations of time include 1 day to 1 month, but the detailed schedule is determined by monitoring liver cholesterol metabolism and plasma levels of LDL and other atherogenic lipoproteins.

The major macrophages involved in liposome clearance are Kupffer cells in the liver and macrophages in bone marrow or spleen. The catabolism in the present invention is a so-called alternative pathway for initiating the conversion of cholesterol to bile acids (macrophages are known to have at least cholesterol catabolism) or sterols (enzymatically modified). has been has or unmodified) other cell (e.g., then a transfer to the liver actual quality) for processing molecules, cholesterol and / or phospholipids to bile and classical bile acid synthesis pathway Including but not limited to direct secretion to.

[0154]   The method also describes herein the control of the effects of cellular senescence.

[0155] The present invention, the oxidation in vitro and in vivo assays, the oxidation-sensitive plasma components assay, and by modified HLD binds to the oxide (oxidative products, and / or or its paraoxonase To assess the efficacy of liposomal therapy by performing assays of its ability to inhibit (via other antioxidant components) and its ability of HDL or plasma or serum or blood to mobilize cholesterol and other exchangeable substances. Including means.

Large liposomes can cause the recruitment of some substances that are also trapped between cells, which is the extracellular region. This extracellular substance causes the following problems: a) When it comes into contact with cells or platelets, they modify their function, b
) Simply take a place.

The rate of cholesterol mobilization can be determined empirically. It is understood that the movement of liposome clearance is different in different species (LUV in mice
t _1/2 is about 8 hours, but about 27 hours in rabbits and longer in humans). Therefore, the calculated velocities may vary from species to species. 300 mg SU
Based on the data on administration of V to rabbits, the peak rate of clearing liposomal cholesterol from plasma is between 3 and 6 hours post injection. At that time,
Liposomes elevate plasma non-esterified cholesterol above just 2 mmol / L; total plasma volume was estimated to be 90 mL in 3 kg rabbits, at which time total liposome cholesterol was 180 μmoles; the t _1/2 for SUV in rabbits is approximately 20 hours and approximately 10%
Are removed in 3 hours; therefore, the peak rate of removal of liposomal cholesterol was approximately 2 μmol / hour / kg, which caused a subsequent increase in plasma cholesterol ester concentrations. The other things to note during the post injection period are:
The rate of liposomal cholesterol removed from plasma was low.
Note also that the liver is the dominant organ for clearance but not the only organ for clearance.

A single injection of 300 mg LUV / kg into 20-22 g mice is calculated to mobilize approximately 2400 nmol cholesterol in the first 24 hours after injection. In contrast to the data with SUVs in rabbits, the first 24 hours of cholesterol mobilization in mice injected with LUVs was very stable. This was calculated to be about 4.7 μmol / hr / kg over this first 24 hours, which was actually greater than the 2 μmol / hr / kg figure, which was the peak rate. This is not a fair comparison. Because the clearance of LUV in mice is 3 times faster than in rabbits. If 4.7 is divided by 3, then 2
Smaller 1.6 μmoles / hour / kg are obtained, which are incomplete estimates.
Human speed can be empirically determined. However, it is clear that LUVs deliver their cholesterol at a stable rate, while SUVs push lipids into the liver simply and quickly.

At body temperature, the most desirable liposomes are fluids within the limits of bilayers, which is called the liquid crystal state. Liposomes in the gel state, which are less fluid, are less desirable.

It is understood that non-esterified cholesterol stimulates macrophages and expresses more tissue factor, a substance known to cause blood clots. This explains the presence of abundant tissue factor in rupture-prone plaques. When ruptured, this plaque-prone plaque exposes tissue factor to plasma, causing a clot that can occlude blood vessels, causing heart failure. This is another example of aberrant cell function, which can be reversed by liposome removal of cholesterol.

[0161] Several human conditions, tissues, cells, characterized by a membrane and / or differential lipid composition of the extracellular region. For example, in atherosclerosis, cholesterol (non-esterified, esterified, and oxidized forms) as well as other lipids accumulated intracellularly and in the arterial wall and other extracellular regions. . These lipids have potentially deleterious biological effects, for example, by altering cell function and by narrowing blood vessel lumens and impeding blood flow. Removal of lipids offers many substantial advantages.
In addition, cells, membranes, tissues and extracellular structures, for example, increase the content and type of antioxidants, reduce the amount of oxidised substances, and the content of substances resistant to oxidization. By increasing the composition of the invention benefit from compositions and modifications that include increasing resistance to oxidative and oxidative damage. During aging, cells have been shown to accumulate sphingomyelin and cholesterol, which alters cell function. These functions can be restored in vitro by removal of these lipids and replacement with phospholipids from the liposomes. A major obstacle to making similar lipid alterations in vitro has been the nature of lipids metabolized from tissues, cells, extracellular regions, and membranes. Natural particles that can metabolize peripheral tissues lipids (e.g., high-density lipoprotein) and synthetic particles (e.g., Sumo Le liposomes) have a considerable disadvantage: they are in a manner that prevents cholesterol homeostasis of the liver, deliver their lipids to the liver, toxic lipoproteins (e.g., low density lipoprotein (LDL), the major atherogenic lipoprotein) results in an increase in the plasma concentration of.

The invention described herein provides methods and compositions relating to the in vivo “reverse” transport of cholesterol and other substances and compounds from peripheral tissues to the liver while controlling plasma LDL levels. provide.

Agarose gel electrophoresis of plasma samples from the last set of rabbits injected with LUV, SUV, or saline (these agarose gels are separated by their charge, which is of a certain type). Particles and different types of particles are not the same). Freshly made SUVs migrate agarose very slowly, indicating that the newly made liposomes have a very small charge. After injection into animals or co-incubation with plasma or lipoproteins, SUVs take up protein from lipoproteins (p
ick up). These proteins give the SUVs more charge and substantially facilitate their migration through the agarose gel. After exposure to plasma, SUVs migrate through these gels faster than LDL.

This gel showed a substantial difference between LUV and SUV. As expected, SUVs migrated before LDL in these gels. However, LUVs migrated almost exactly to where the newly prepared liposomes without protein migrate. This result indicates that LUVs, unlike SUVs, do not readily uptake proteins from circulating lipoproteins.

There is direct proof of this difference between liposomes. Human HDL, which has the majority of proteins incorporated by liposomes, is incubated with either LUV or SUV, then the liposomes are isolated again and their protein to phospholipid ratio assayed. SU per amount of liposome phospholipid
V picks up about 40 times more protein than LUV. This difference, of surface curvature difference is so that occur because: towards SUV is small, as a result, their surface is more sharply curved, thus more is distorted large crisis, resulting protein is more easily Can be inserted into.

There are two most likely metabolic effects of the difference in protein uptake between the two types of liposomes, and are: VLDL has two metabolic fate: can it be removed from plasma before it is completely converted to LDL by a lipolytic enzyme, or this LDL
Can be completely converted to circulating LDL. SUVs remove apoE from VLDL, thereby slowing its clearance from plasma and helping its conversion to LDL. In contrast, LUV leaves apoE in VLDL, resulting in no increase in plasma LDL concentration.

2. Absorbed apoprotein causes liposomes to differentiate into different liver metabolism pools and / or
Or it may play a role in directing to different speeds.

Here are several methods for assaying the effect on oxidation in vivo: Catella F, Reilly MP, Delanty N, La.
wson JA, Moran N, Meagher E, FitzGerald
GA, Physiologic formation of 8-epi
-PGF2 alpha in vivo is not affected
by cyclooxygenase inhibition. Adv Pro
stalandin Thromboxane Leukot Res. 23
: 233-236, 1995. These authors are the end products of lipid oxidation,
8-epi-PGF ₂ α is described. They propose that this molecule can be used as a measure of lipid oxidative flux in animals. This is the antioxidant level (
Like dietary influences), thiobarbituric acid-reactive substances (some sugars interfere with this assay), and short-lived oxidation intermediates (which do not show the total flux of the substance being oxidized). However, it is superior to other commonly used in vivo measurements of oxidation. By removing oxidised lipids from the periphery, administration of LUVs results in less total oxidative flux in vivo, 8-epi-PGF ₂
α is a suitable method to measure this lesser total oxidative flux; Ca
det J, Ravanat JL, Buchko GW, Yeo HC, Am
es BN, Singlet xygen DNA damage: chro
matographic and mass spectrometric a
analysis of damage products. Methods E
nzymol. 234: 79-88, 1994. These describe the final product of DNA oxidation, 8-oxo-2'-deoxyguanosine. as mentioned above,
This molecule can be used as a measure of DNA oxidative flux in animals. L
Administration of UV reduces the in vivo DNA oxidative flux, which is due to this D
A suitable method for measuring NA oxidative flux; and Xia E, R
ao G, Van Remmen H, Heydari AR, Richard
son A, Activities of antioxidant enzy
mes in various tissue of male Fisher
r 344 rats are alternated by food restr
motion. J Nutr. 125 (2): 195-201, 1995. Antioxidant enzymes in tissues are measured to show their deoxidizing capacity. LUV helps this. Antioxidant levels (vitamin E, ascorbate, urate); oxidized and reduced glutathione; and many other measurements can be used to assess peripheral oxidative and oxidative damage. These and other measures are also combined with LUV administration to assess the efficacy of treatment.

Other particles that mimic those properties of large liposomes act similarly, metabolizing and exchanging peripheral lipids and other exchangeable substances while avoiding deleterious disruption in liver cholesterol homeostasis. Deliver the substance. For example, they are compositions and structures that are slowly taken up by the liver and / or liver and endothelial windows of compositions and structures that do not readily acquire certain endogenous proteins.
It contains two large emulsion particles in order to penetrate the trae). Such emulsions can be made with or without proteins and can be made from phospholipids and neutral lipids such as triglycerides or another neutral lipid.

The present invention also provides a pharmaceutical composition comprising or consisting essentially of sized liposomes and compositions such that the liposomes are slowly taken up by the liver.

The present invention also includes a method of reverse transporting cholesterol from peripheral tissues to the liver in vivo while controlling plasma LDL concentration, which method comprises converting phospholipids substantially free of sterols during the treatment period. It comprises the step of transdermally administering a therapeutically effective amount of a large variety of large liposomes, whereby the liposomes take up cholesterol during the treatment period. This method involves the optional step of enhancing the tissue penetration of cholesterol acceptors and increasing the extraction of tissue cholesterol and other exchangeable substances by co-administration of effective amounts of the compounds. This compound is selected from the group consisting of small cholesterol acceptors and agents that increase small endogenous cholesterol acceptors. In a variant, co-administration of this compound is concomitant with parenteral administration of large liposomes. In another modification , co-administration of compounds is separated from therapeutically effective amounts of parenteral administration of various large liposomes at just the right and effective time. Effective times range from about 1 minute to about 2 weeks.

In another aspect, the invention includes an improved method of reducing the lipid content of arterial injury, which method comprises administering a therapeutically effective amount of an agent to a subject from peripheral tissues in vivo. Inducing reverse transport of cholesterol to the liver.
The agent is selected from the group consisting of large liposomes composed of sterols and phospholipids substantially free of small acceptors; the subject's plasma LDL concentration is periodically monitored to obtain an LDL concentration profile; A therapeutically effective amount of the agent is adjusted in response to the concentration profile; and the pharmaceutical agent is administered to the subject. The agent is selected from the group of compounds for lowering LDL concentration, small acceptor, and compositions that increase HDL concentration in response to an LDL concentration profile, which results in a reduction in lipid content of arterial injury.
Effectively treated and monitored over the treatment period. Arterial injury includes lipid-rich, rupture-prone type IV and type V arterial injury. Puncture rupture, thrombosis, and tissue infarction are greatly reduced.

In yet another aspect, the invention provides an improved method of assessing the efficiency of a treatment for reducing the lipid content of an arterial injury, which injury contacts plasma and its components. The method comprises inducing reverse transport of cholesterol from peripheral tissues to the liver in vivo by administering to the subject a therapeutically effective amount of the drug. The drug is selected from the group consisting of large liposomes composed of sterols and phospholipids substantially free of small acceptors; and plasma components are periodically monitored by the assay. This assay is for plasma non-esterified cholesterol and phospholipids, fecal bile acids and cholesterol assays, bile bile acids and cholesterol assays, liver gene expression assays in liver biopsies, peripheral Assay of gene expression in blood leukocytes (this gene includes genes involved in cholesterol metabolism)
, Plasma LDL concentration assay, and vascular imaging techniques. Vascular imaging techniques include cardiac catheterization, magnetic resonance imaging, ultrasound, ultrafast CT, radionuclide assays (including stress-thallium scans where appropriate), any perfusion assay (eg PET scan), and ECHO. Selected from the group consisting of any functional assay such as

The invention also includes a method of beneficially altering arterial function, platelet function and controlling plasma LDL levels and hepatic cholesterol homeostasis in vivo, which method comprises:
Administering parenterally a therapeutically effective amount of a variety of large liposomes composed of phospholipids that are substantially free of sterols for the duration of the treatment, with or without the administration of other agents. Include. Other agents optionally include small acceptors and agents that reduce LDL. Optionally, the method includes the step of making a measurement of arterial function. This measurement includes blood flow, oxygen delivery, measurement of endothelial-induced relaxation factor, measurement of intracellular calcium concentration in arterial cells, measurement of arterial cell proliferation, assay of arterial enzyme, assay in the presence of calcium channel blocker, It is selected from the group consisting of arterial uptake, accumulation and maintenance assays of lipid proteins, liposomal arterial accumulation assays, liposomal arterial maintenance assays, gene product assays, and arterial cell function assays. The measurement of endothelium-induced relaxation factor is selected from the group consisting of a functional determination of endothelium-dependent arterial relaxation, a chemical determination of the production of endothelial relaxation factor, and an assay for nitric oxide synthase.

[0175] Plasma LDL levels while beneficially modify platelet function, arterial function, a method for controlling a platelet function in liver storehouse cholesterol <br/> roll homeostasis and in vivo are also included. This method involves parenterally administering a therapeutically effective amount of a large number of large liposomes , ie liposomes administered with or without other agents, consisting of phospholipids substantially free of sterols during the treatment. The step of performing is included. This method optionally includes the step of measuring arterial function. The measurement is selected from the group consisting of measurement of endothelial-induced relaxation factor, measurement of intracellular calcium concentration in arterial cells, measurement of arterial cell proliferation, assay of arterial enzyme, and assay of gene product. The measurement of endothelial relaxant is selected from the group consisting of functional determination of endothelium-dependent arterial relaxation and chemical determination of endothelial relaxant production.

[0176] vivo macrophages (e.g., hepatic built macrophages) affects how also included in the plasma component or structural aspects of decomposing and arterial cholesterol using, comprising administering a therapeutically effective amount to a subject Of liposomes, the liposomes being substantially cholesterol-free and of a size and composition such that the liposomes can be taken up by and degraded by macrophages. Cholesterol is recruited by liposomes, resulting in liposomes that are taken up and degraded by macrophages. The method can also include the step of periodically monitoring plasma components using the assay. This assay, plasma unesterified cholesterol and assays for phospholipids, assay of plasma cholesterol ester moving protein activity, bile acids and cholesterol assays in stool, an assay of hepatic warehouse gene expression in the liver Biopushi, in peripheral binding leukocytes It is selected from the group consisting of gene expression assays, which include genes involved in cholesterol metabolism, plasma LDL concentration assays and vascular imaging techniques.

In yet another aspect, the invention provides a method of delivering a drug in vivo, a method of preventing the deleterious division of liver storage cholesterol homeostasis, including the step of drug trapping. including. This agent is selected cholesterol small liposomes without cholesterol liposomes, emulsions, liver real quality to result liposomes ingested slowly initially, from the group consisting of liver real quality to result an emulsion ingested slowly initially . The drug is selected from the group consisting of drugs with proteins and drugs without proteins to obtain trapped drugs. The method also includes administering a therapeutically effective amount of drug trapped during the treatment period. This administering step comprises slowly infecting the trapped drug. A multifaceted, step of administering comprises the steps of in order to prevent harmful disruption in liver storehouse cholesterol homeostasis, approximately separate number, comprising administering a small dose of the drug, and using low doses of the drug encompasses, whereby is possible to divide the liver storehouse cholesterol homeostasis, it is prevented.

[0178] Plasma LDL levels, hepatic storehouse cholesterol homeostasis, arterial enzymes, arterial function, and methods to modify the method and plasma plate hormone production for controlling platelet function also included. The method comprises the step of parenterally administering a therapeutically effective amount of a large number of large liposomes comprised of phospholipids substantially free of sterols during the treatment period. The effective amount is administered in a dosage, and the dosage is selected from single and repeated doses. The method comprises the steps of diagnosing the efficacy of the administration by optionally measuring hormone production and adjusting the effective amount in response to this measurement. The measurement of hormone production is an assay selected from the group consisting of an assay for thromboxane, an assay for prostacyclin, an assay for prostaglandins, an assay for leukotrienes, and an assay for their derivatives.

[0179]   In a still further aspect, the present invention increases plasma HDL levels while increasing plasma
LDL level, liverWarehouseCholesterol homeostasis, and liverWarehouseControl gene expression
Provide a way to control. This method involves parenterally administering a therapeutically effective amount of a first drug.
Including the step of This first drug is often used to induce HDL levels during the treatment period.
Number ofSmall liposomesContains. This method then administers the same second drug.
Including the process. This second drug is a phospholipid that is substantially free of sterols during the treatment period.
Consists ofLarge liposomeincluding. This effective dose can be single dose and repeated
It is administered in a dosage selected from doses. Co-administrationSmall liposomesIs the liver Warehouse Stimulating deleterious changes in cholesterol homeostasis and plasma
Acts to prevent an increase in LDL.In modification, This first drug is
, EssentiallySmall liposomesAnd this second drug consists essentially ofRa Diliposome Consists of. This method also uses plasma HD before, during and after the treatment period.
Diagnosing the efficacy of administration by making measurements of L and LDL levels
including.

[0180] Plasma LDL levels, while controlling the hepatic storehouse cholesterol homeostasis in vivo, the method changes the cell membrane composition and function, are also described herein. The method comprises the step of parenterally administering a therapeutically effective amount of a plurality of large liposomes composed of phospholipids substantially free of sterols during the treatment period. This effective amount is administered in a dosage selected from single and repeated doses. The method comprises the step of co-administering a cholesterol small acceptor, acceptor sphingomyelin, small again acceptor selected from the group consisting of an acceptor of the acceptor and lipid lysophosphatidylcholine. This method measures membrane fluidity, transmembrane ion flux (the ion is selected from the group consisting of calcium, sodium, and potassium ions), membrane fragility assay, and membrane function assay. It may optionally include the step of diagnosing the efficacy of administration by performing a measurement selected from the group consisting of.

In a further embodiment, the present invention is for treating angina entering the liver of a subject consisting essentially of liposomes selected from the group of unilamellar vesicles, multilamellar vesicles, combinations thereof and derivatives thereof. Pharmaceutical composition of.
These particles are selected from the group of particles substantially free of cholesterol and particles free of cholesterol.

The non-liposome particles are selected from the group consisting of triglyceride phospholipid emulsions. As the emulsion, the emulsion is not taken into the liver actual quality result quickly, the actual quality therefore not taken long term emulsion and triglycerides - phospholipid - protein emulsions.

Also included in the invention is a pharmaceutical composition for reducing the size of arterial lesions that enter the liver of a subject consisting essentially of the drug trapped within the drug. Select this drug, cholesterol small liposomes without cholesterol liposomes, emulsions, from the group consisting of liver real quality to thus slow the liposome is taken initially, and liver real quality to result an emulsion which is slowly consumed initially To be done. The drug is selected from the group consisting of drug with protein and drug without protein.

[0184] The present invention also provides, while increasing plasma HDL concentrations, plasma LDL levels, and provides a pharmaceutical composition for controlling the hepatic built co <br/> less Te rolls homeostasis, and hepatic built gene expression, the composition comprises a second agent comprising a large liposome comprised of phospholipids first agent and sterol substantially free comprise a plurality of Sumoruri liposomes. Used to raise HDL concentrations.

[0185] In yet another aspect, cells from Kupffer cells in the real Shitsuma - a method of controlling cholesterol metabolism in the liver actual quality in a subject in vivo through cell communication is included. The method involves administering to the subject a liposome composition. The liposome composition is selected from the group consisting of large unilamellar vesicles and large multilamellar vesicles. The liposomes have an average diameter of about 100-180 nanometers. LDL levels in the subject do not increase. This method also
Diagnosing the efficacy of controlling cholesterol metabolism by assaying an indicator in the subject. This indicator is the subject's plasma L
DL concentrations, liver storehouse gene expression in a subject, sterol excretion controlling the definitive cholesterol metabolism in the liver actual quality in a subject, and is selected from the group consisting of excretion of sterol in the bile of the subject; and administration in response to the assay Adjust.

The present invention further provides modalities of manipulation of atherogenic lipoproteins, cellular and extracellular structures modified by the compositions described herein that result in a beneficial physiological effect.

Diagnosis or conditions generally associated with endothelial function or dysfunction can be treated using the methods disclosed herein. By way of example, some of these conditions or diseases include hypertension, epilepsy, hyperviscosity syndrome, preeclampsia, and inflammation. Inflammation includes autoimmune and non-autoimmune conditions.

The methods of rapidly and substantially treating angina disclosed herein provide for at least three synergistic beneficial effects of liposome treatment (eg, large "empty" phospholipid vehicle- "LEV" -wherein , "Empty" refers to any conventional term indicating that the encapsulated drug is not essential). These three beneficial effects include (1) recovery of endothelial dysfunction, (2) recovery of platelet hyperactivity, and (3) reduction of whole blood viscosity. All three of these are relatively quick and substantial treatment effects. These effects are particularly important when there are occlusive, fibroblastic, lipid-poor arterial lesions that are unlikely to disappear rapidly or substantially with alterations in lipid transport. In the case of the heart, the in vivo method is
Helps relieve angina and its associated signs and symptoms such as shortened breathing, decreased exercise tolerance, heart wall dyskinesias, arrhythmias, and cardiac function in general. In the case of the brain, the treatments disclosed herein help alleviate transient cerebral ischemic attacks and their associated signs and symptoms (eg, neurological dysfunction). In the case of the lower extremities, the treatments disclosed herein ameliorate lameness and its associated signs and symptoms. Developmental organ perfusion and function are also assisted.

The intravenous route of administration may also be premixed prior to the time of simultaneous co-administration,
Or co-administration with red blood cells and other blood products, either immediately before or immediately after the administration of blood products (up to a week). Therefore, the present invention also contemplates the use of liposomes with blood transfusion products.

The approaches described herein are directed to other therapeutic agents directed at the arterial wall (vasodilators, agents that interfere with cell adhesion molecules, anti-inflammatory agents, agents that modify cytokines, antioxidant treatments). Drugs and inhibitors of arterial wall enzymes (eg acetyl CoA:
(Including cholesterol acyltransferase, lipase, myeloperoxidase, lipoxygenase and phospholipase)). It is contemplated that the use of liposomes herein results in a synergistic effect with each of these compounds, the others described herein, and in part the therapeutic agents. Because liposomes is because act by mechanisms different from the mechanisms used by these therapeutic agents.

Various cardiovascular agents are also used in the methods of the invention. Examples of these drugs include blood modifiers, anticoagulants, antiplatelet agents, thrombolytic agents, and adrenergic blockers.
er), an adrenergic stimulant, an α / β adrenergic blocker, an angiotensin converting enzyme (ACE) inhibitor (for example, Quinapril (Acc
upril®, Parke-Davis), ramipril (Altac)
e®, Hoechst), Captopril, Benazepril (Loten)
sin (registered trademark), Novartis, trandolapril (Mavik (registered trademark), Knoll), fosinopril (Monopril (registered trademark), Br)
istol-Myers), lisinopril (Prinivil®, M
erck), moexipril (Univasc®, Schwarz)
, Enalapril (Vasotec® tablets, Merck), enaprilat (Vasotec®, iv, Merck), lisinopril (Z
estril®, Zeneca), its active metabolites, and / or its derivatives.

ACE inhibitors with calcium channel blockers and ACE inhibitors with diuretics can also be used with the present invention.

Angiotensin II receptor antagonists, including selective AT-I subtype angiotensin II receptor antagonists, also find use with the present invention (eg, Candesartan cilexetil (Ataca).
nd (registered trademark), Astra), Irbesartan (Avapro (registered trademark), Bristol-Myers Squibb or Sanofi), L
osartan (Cozaa®, Merck), Valsarta
n (Diovan®, Novartis), angiotensin II receptor antagonist with diuretics, its active metabolites, and / or its derivatives).

[0194]   Group I-IV antiarrhythmic agents are also used in the present invention, Group I antiarrhythmic agents.
Examples include the following: Cardioquin^TM(The common name is
It is a nisin), Ethmozine^TM, Mexitil^TM, Norspace^TM (Generic name is disopyramide), Procanbid^TM(General name is Proca
Inamide), Quiniaglute^TM, Quinindex^TM, Ryth
mol^TM, Tambocor^TM, And Tonocard^TM. Group II antiarrhythmic drugs
Examples include the following: Betaspace^TM(General name is Sotaro
), Brevibloc^TM(Generic name is esmolol), In
general^TM(Common name is propranolol), and Special^TM (The common name is Acebutrol). Examples of the group III antiarrhythmic drug are as follows.
The following are included: Betaspace^TM, Cordarone^TM, Covert ^TM And Pacerone^TM. Examples of group IV antiarrhythmic drugs include the following:
Is: Calan^TM(Common name is verapamil) and Cardizem ^TM (The common name is diltiazem). Various anti-arrhythmias used in the present invention
As vascular drugs, Adenocard (generic name is adenosine), Lano
xicaps^TM(Common name is digoxin), and Lanoxin^TM(one
The common name is digoxin).

Antilipidemic agent used in the present invention
nt) includes bile acid isolates, Fibric Acid derivatives, HMG-CoA reductase inhibitors, and nicotinic acid.

Other agents used in the present invention include β-adrenergic blockers, β-adrenergic blockers with diuretics, calcium channel blockers, diuretics, (
For example, Mechiroshin (carbonic dehydrogenase inhibitors, combination (combinat
Ion) diuretics, loop diuretics, potassium-sparing diuretics, and thiazides and related diuretics), hypertensive emergency drugs, muscle contractors, various cardiovascular drugs (eg, Metyrosine (Demser ^™ , Merck), mecamylamine (Inversin).
e ^™ , Merck), Fentolamine (Regitine ^™ , Novart
is), Abciximab (Reopro ^™ , Centocor & Lil
ly) rauwolfia derivatives and combinations), vasodilators, coronary vasodilators and peripheral vasodilators and combinations, and vasoconstrictors.

The invention described herein also provides a method of treatment of vascular dysfunction. The vascular dysfunction, dysfunction of the resistance vessels, myocardial dysfunction in arterioles, dysfunction conductive vessels, and epicardial coronary insufficiency include, but are not limited to.

Equivalents to the various angina discussed herein (ie, the effects of transient myocardial ischemia associated with standard angina (eg, substernal discomfort; lethargy, tightness, tightening). , Breathing (smothering), or choking
), By way of example, include: mechanical, biochemical, and electrical dysfunction of the myocardium, failure of normal muscle relaxation and contraction, transient left ventricular insufficiency (a typical manifestation of this is , Shortness of breathing or decreased exercise tolerance), papillary muscle failure (including mitral regurgitation, ventricular contractile defocusing (eg, causing segmental distension or dyskinesia) and / or Extremely diminished efficiency of myocardial pump function, wide range of abnormalities in cardiomyocyte metabolism, function, and structure, inability to oxidize fatty acids, glucose to lactate conversion, (
Intracellular pH and high energy phosphate (adenosine triphosphate (A
TP), and creatine phosphate) resulting in reduced myocardial storage), defective cell membrane function, resulting in potassium leakage and sodium uptake by myocytes),
Characteristic electrocardiographic changes (e.g., by inverting the T-wave, and later as evidenced by replacement of the ST-ku, repolarization abnormalities), electrical instability (which is ventricular tachycardia or ventricular fibrillation Sudden death as a result of ischemia-induced malignant ventricular arrhythmia, other areas (eg, left shoulder, arms, forearm and hand ulnar surface, back, neck, jaw, teeth, and / or epigastrium) Dissipated in the.

The present invention also has beneficial effects in treating conditions that affect vascular anatomy. Vascular anatomical structures include, by way of example: vascular vessels,
Posterior capillary venules, venules, arterioles, capillaries, cavernous
sineous), arteries, veins, any vasculature that normally lines the endothelium, and / or any vasculature that normally lines the endothelium but is denutrified.

The invention disclosed herein is also understood to include treatment of a wide range of cholesterol metabolism, lipid metabolism, and / or aging related conditions. Further exemplary conditions include cancer and predisposition to cancer (eg, colon cancer (which also
(Can be prevented with cholesterol synthesis inhibitor aspirin or related compounds)
, And breast cancer); alopecia (including male pattern baldness); skin wrinkles (including prevention and reversal of wrinkles); gray hair (including premature graying); chemical or radiotoxicity (eg, , Derived from the removal of lipophilic toxins such as oxidative products); and conditions associated with the signs or symptoms of accelerated aging. Treatment is applied to human and non-human animals, including pets.

The treatments, compositions, and methods disclosed herein are also useful in treating Tangier disease and related conditions. Tangier disease, a genetic disease in which patients have abnormally low levels of plasma high density lipoprotein cholesterol with enlarged and yellowed tonsils and spleen, encodes the protein ATP-cassette binding protein 1 (ABC1) It has recently been discovered that it is associated with a deficiency in the enclade gene. ABC1 binds to intracellular cholesterol, and the cholesterol has been shown to transport to the cell membrane receptor responsible for transporting cholesterol to extracellular acceptor. Cells with ABC1 deficiency are unable to release cholesterol, even in the presence of excess extracellular acceptor. In contrast, cells with excess levels of ABC1 release a lot of cholesterol to extracellular acceptors, thus killing cells with cholesterol deficiency.

ABC1 is the first intracellular cholesterol transport protein identified to play a role in the first step of the reverse cholesterol transport process. Identification of this protein has created the opportunity for the identification of drugs that stimulate this process and enhance intracellular transfer of cholesterol to the cell membrane. At this point, the rate at which cells can eventually release cholesterol depends on the presence of sufficient extracellular acceptor molecules. The present invention is also useful in combination with drugs that enhance intracellular transfer of cholesterol to the cell membrane.

HDL, and in particular HDL1, is known to act as an acceptor of cellular cholesterol. However, HDL has a limited capacity to acquire cholesterol and acts as a more efficient acceptor of cholesterol in the presence of other molecules that can act as cholesterol reservoirs or sinks. A particularly efficient and cooperative way to enhance cellular cholesterol efflux is by the combination of HDL and empty phospholipid liposomes as described herein. In this situation, small HDL molecule cholesterol from cell membranes shea Yatoru, and transport of cholesterol to the liposomes. Liposomes have a larger capacity related to quite large, and cholesterol. Then HD
L is free to bind more cellular cholesterol and continues the shuttle function. In this situation, limited amounts of HDL lipoproteins may facilitate the removal of even more cellular cholesterol.

When the deficiency in ABC1 limits the cell's ability to transport cholesterol to the cell membrane, extracellular acceptor capacity is by no means a rate-limiting step in the cholesterol efflux process. However, in the presence of pharmaceutical agents that stimulate intracellular transport of cholesterol by AbC1 or other intracellular cholesterol transport proteins, the ability of HDL to accept all translocated cholesterol may be a limiting factor in the reverse cholesterol transport process. .

Accordingly, the present invention provides that the vesicles are hepatically fenestral.
Empty phospholipid vesicles with a sufficiently large mean diameter to exclude passage through (which is an aperture of about 100 nm), combined with a drug that stimulates ABC1 function or a similar intracellular cholesterol transport protein, Requires intravenous administration. Of course, other liposome compositions described herein can also be used. As a result of the combination, the extracellular cholesterol acceptor particles have an enhanced capacity for cellular cholesterol. The size of the phospholipid liposomes acceptor particles prevents the uptake of cholesterol efflux hepatic built substantially. As a result, avoid cholesterol interferes with the liver built-cholesterol homeostasis. Instead, these particles are cleared by Kupffer cells, where cholesterol is finally removed in bile.

The present invention is particularly useful in treating patients suffering from any number of genetic conditions that produce very low levels of HDL cholesterol. Many of these conditions are
Makes patients susceptible to accelerated atherosclerosis and heart disease.

Although only a few preferred embodiments of the invention have been described herein above, it is understood that the embodiments may be modified and altered without departing from the central spirit and scope of the invention, Those skilled in the art will understand. Accordingly, the preferred embodiments described above herein are to be considered in all respects as illustrative and not restrictive,
The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all variations that come within the meaning and range of equivalency of the claims are to be embraced herein. Is intended.

[Brief description of drawings]

[Figure 1]   FIG. 1 is a side cross-section of lipoproteins and liposomes.

FIG. 2 shows liver mRNA levels (pg / pg for CETP, HMG-CoAR, LDL receptor, and 7a hydroxylase; and LDL ChE.
) Table is illustrated.

FIG. 3 illustrates plasma LDL cholesterol ester concentration in response to LUV, SUV or saline injection over time in one modification.

FIG. 4 illustrates plasma LDL cholesterol ester concentration in response to LUV, SUV or saline injection over time in one modification.

FIG. 5 illustrates LDL receptor mRNA levels in the liver in response to LUV, SUV or saline injections over time.

FIG. 6. H in liver in response to injection of LUV, SUV or saline.
3 illustrates MG-CoA reductase mRNA levels.

FIG. 7 illustrates cholesterol transesterification protein mRNA levels in the liver in response to LUV, SUV or saline injections.

FIG. 8: 7 in liver in response to injection of LUV, SUV or saline.
-Exemplifies alpha hydroxylase mRNA levels.

[Figure 9]   FIG. 9 illustrates important points regarding LUV and arteriosclerosis.

FIG. 10 illustrates plasma LDL non-esterified cholesterol levels in response to LUV, SUV or saline injections over time.

FIG. 11 illustrates plasma LDL esterified cholesterol concentrations in response to LUV, SUV or saline injections over time.

FIG. 12 illustrates LDL esterified cholesterol levels in response to LUV, SUV or saline injections.

FIG. 13: Plasma VLDL in response to LUV, SUV or saline injections.
The esterified cholesterol concentration is exemplified.

Figure 14, LUV, in response to injection of SUV or saline illustrate H DL esterified cholesterol concentrations.

Figure 15, LUV, in response to injection of SUV or saline illustrate H DL esterified cholesterol concentrations.

FIG. 16 shows LUVs in control mice or apoE knockout mice.
3 illustrates the time course of cholesterol transfer after injection.

Figure 17 illustrates the time course of LUV clearance in control mice and apo E mice.

FIG. 18 illustrates that the compositions and methods of the present invention are effective in humans.

FIG. 19 shows a perspective view of the improved hemodialysis system of the present invention and an improved method of hemodialysis.

FIG. 20 illustrates a perspective view of an improved peritoneal dialysis system 2000 and a perspective view of a method of peritoneal dialysis.

FIG. 21   FIG. 21 shows a modified 2100 modified peritoneal dialysis system with assay means. Perspective view of And illustrates the method of peritoneal dialysis and analysis of the liquid used.

FIG. 22 illustrates an improved cardiac catheterization and / or angioplasty system 22.
00 is a perspective view and a method of cardiac catheterization and / or angioplasty.

FIG. 23 illustrates a perspective view of a modified cardiac catheterization and / or angioplasty system modification 2300 and a method of cardiac catheterization and / or angioplasty.

FIG. 24 illustrates a graph of liver fat content in response to LUV, SUV or saline injections.

FIG. 25 illustrates plasma free cholesterol concentration after repeated injections of SUV or LUV (300 mg / kg) into NZW rabbits.

FIG. 26 illustrates plasma cholesterol ester concentration after repeated injections of SUV or LUV (300 mg / kg) into NZW rabbits.

FIG. 27   FIG. 27 illustrates the changes in plasma components after repeated injections of SUV.

FIG. 28 illustrates an agarose gel electropherogram of whole plasma after repeated injections of LUV, SUV or saline. (Reason for correction 1: Regarding the scope of claims) The scope of claims in the translation submitted on November 14, 2001 will be amended. This is a correction that can be applied even with general correction. (Reason for correction 2: Detailed explanation of the invention and brief explanation of the drawings) The detailed explanation of the invention and the brief explanation of the drawings in the translated text submitted on November 14, 2001 are all mistranslated. There was, so we will correct the mistranslation to change to an appropriate translation.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 9/70 401 A61K 9/70 401 31/138 31/138 31/165 31/165 31/166 31 / 166 31/167 31/167 31/18 31/18 31/21 31/21 31/216 31/216 31/277 31/277 31/40 31/40 31/401 31/401 31/404 31/404 31 / 4402 31/4402 31/4422 31/4422 31/4439 31/4439 31/4458 31/4458 31/4704 31/4704 31/49 31/49 31/5377 31/5377 31/55 31/55 31/675 31/675 31/7048 31/7048 31/7076 31/7076 38/55 45/00 45/00 47/30 47/30 47/34 47/34 A61P 3/00 A61P 3/00 5/16 5/16 7/02 7/02 7/06 7/06 9/04 9/04 9/06 9/06 9/10 9/10 9/12 9/12 11/00 11/00 23/00 23/00 25 / 20 25/20 G01N 33/53 W G01N 33/53 33/543 585 33/543 585 587 587 A61K 37/64 (81) Designated country EP (AT, BE, CH, CY, DE, DK) ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES. , FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Williams, Kevin John USA Pennsylvania 19096 , Wine Wood, Wistar Road 425 F Term (Reference) 4C076 AA06 AA19 AA20 AA21 AA72 AA93 BB01 BB02 BB22 BB27 BB31 CC11 CC13 CC14 EE01 EE27 4C084 BC19 BC01 BA14 BA23 DC40 MA02 MA24 ZA36 ZA42 A01A21 A02 BC28 BC32 BC71 BC85 CB17 DA38 DA41 DA42 EA10 EA18 GA10 GA12 MA01 MA02 MA03 MA04 MA05 MA13 MA24 MA32 MA52 MA57 MA63 NA14 ZA36 ZA42 ZA45 ZA54 ZA89 ZC20 4C206 AA01 AA02 DB17 DB20 DB43 EA06 EA07 GA21 MA11 GA11 FA11 FA18 FA19 FA19 FA19 FA19 FA19 FA19 FA19 FA19 FA19 FA19 FA19 FA11 FA19 FA19 FA11 FA19 FA11 FA19 FA11 MA04 MA05 MA33 MA44 MA52 MA72 MA77 MA83 NA14 ZA36 ZA42 ZA54 ZA89 ZC20

Claims (31)

[Claims] 1. A composition for use in the treatment of angina and / or angina equivalents or lameness, wherein the composition comprises a therapeutically effective amount of liposomes comprising a phospholipid given to a subject during the treatment period. A composition comprising. 2. The composition of claim 1, wherein the angina is selected from the group consisting of stable angina, unstable angina and variant angina. 3. The composition of claim 1, wherein the angina is angina. 4. The liposome, wherein the liposome has a size and shape larger than the perforation of endothelium sinusoids of the hepatic lining in the liver, a large liposome that does not substantially increase the concentration of plasma LDL, and a plasma atherogenic lipo. Selected from the group consisting of large liposomes that do not substantially increase the concentration of protein,
The composition of claim 1. 5. The therapeutically effective amount is about 10 per kg body weight per dose.
The composition of claim 1, wherein the composition is in the range of mg to about 1600 mg of phospholipid. 6. The composition according to claim 1, wherein the liposome is selected from the group consisting of unilamellar vesicles, small lamellar vesicles and multilamellar vesicles. 7. The liposomes have a mean diameter greater than about 50 nm, liposomes having a mean diameter greater than about 80 nm, about 100 nm.
Liposomes having a larger average diameter, liposomes having an average diameter in the range of about 100 nm to about 150 nm, liposomes having an average diameter in the range of about 150 nm to about 200 nm, average diameters in the range between about 200 nm and about 300 nm. The composition of claim 1 selected from the group consisting of liposomes having, average liposomes having an average diameter in the range of about 250 nm to 400 nm, and combinations thereof. 8. A kit comprising the composition of claim 1, wherein the kit further comprises an effective amount of an anti-angina drug other than the liposome. 9. The anti-angina drug is selected from the group consisting of nitrates, β blockers, calcium channel antagonists, coronary vasodilators, lipid lowering agents, post-load reducing agents, inotropic agents, pre-load reducing agents and opiates. The kit according to claim 8, which is: 10. The nitrate comprises nitroglycerin, sublingually administered nitroglycerin, long-acting nitrate, sublingual nitrate preparation, buccal nitrate preparation, oral nitrate preparation, spray nitrate preparation, Oral nitroglycerin spray, isosorbide dinitrate preparation, isosorbide-5-
Mononitrate preparation, non-sublingual nitrate preparation, sustained release preparation of isosorbide-5-mononitrate, topical nitroglycerin, nitroglycerin ointment, nitroglycerin containing transdermal patch, and nitroglycerin impregnated silicone gel or polymer The kit according to claim 9, which is selected from the group consisting of matrices. 11. The β-blocker is a non-selective β-blocker, propranolol,
Blocks nadolol, penbutolol, pindolol, sotalol, timolol, carteolol, drugs that inhibit both β1 and β2 receptors, cardioselective β blockers, acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and β1 receptor 10. The kit of claim 9, wherein is selected from the group consisting of drugs that have less effect on the β2 receptor. 12. The calcium channel antagonist, a compound that inhibits the movement of calcium ions through slow channels in the membranes of myocardium and smooth muscle by non-competitive blockade of calcium antagonists, voltage-sensitive L-type calcium channels, dihydropyridine, nifedipine ( (Registered trademark), phenylalkylamine, verapamil (registered trademark), benzothiazepine, dil
tiazem (registered trademark), nicardipine (registered trademark), amlod
ipine® and bepridil®, second generation calcium antagonists, nicardiopine®, isradii
pine (registered trademark), amlodipine (registered trademark), felodipi
selected from the group consisting of ne (registered trademark) and dihydropyridine derivatives,
The kit according to claim 9. 13. The angiotensin converting enzyme (ACE) inhibitor is quinapril, Accupril®, ramipril, Altace®, captopril, benazepril, Lotensin®, trandolapril, Mavik®. , Hosinopril, Monopril®, Lysinopril, Prinivir®, Moexipril, U
The kit according to claim 9, which is selected from the group consisting of nivasc (registered trademark), enalapril, Vasotec (registered trademark), enalaprilato, lisinopril, Zestril (registered trademark), active metabolites thereof, and derivatives thereof. 14. The antiarrhythmic drug is quinidine, etomozine, m.
exitil, disopyranide, procainamide, quinaglute, qu
index, rythmol, tambocor, tonocard, sotalol, esmolol, propranolol, acebutolol, betaspace,
The kit according to claim 9, which is selected from the group consisting of cordarone, covert, calan, verapamil, diltiazem, adenosine, digoxin, active metabolites thereof, derivatives thereof and combinations thereof. 15. A kit comprising the composition of claim 1, further comprising administering an angiotensin converting enzyme (ACE) inhibitor to the subject. 16. The kit of claim 15, wherein the kit further comprises a device for the treatment of arrhythmias comprising an antiarrhythmic drug and / or AICD to the subject. 17. A kit comprising the composition of claim 1, further comprising an antithrombotic therapy. 18. The antithrombotic treatment comprises administering a therapeutically effective amount of an antiplatelet drug, administering a therapeutically effective amount of a drug that interferes with fibrin clot formation, and administering a therapeutically effective amount of a thrombolytic therapy. 18. The kit of claim 17, selected from the group consisting of: 19. The atherogenic lipoprotein is LDL, Lp (a),
A kit comprising the composition of claim 1 selected from the group consisting of lipoproteins including apo-B, VLDL, β-VLDL, and remnant lipoprotein. 20. The composition of claim 1, wherein the anti-angina therapy further comprises treatment of a coexisting worsening condition, wherein treatment of the coexisting worsening condition is treatment of hypertension, thyroid function. It is selected from the group consisting of treatment of hyperactivity, treatment of lung disease, treatment of heart failure, treatment of hypercatabolism, and treatment of anemia. 21. Use of the composition according to claim 1, wherein the level of plasma atherogenic lipoproteins is controlled or monitored. 22. The use of a composition according to claim 1, wherein the angina is selected from the group consisting of stable angina, unstable angina and variant angina. 23. Use of a composition according to claim 1, wherein the angina is angina. 24. The use of a composition according to claim 1, wherein the use comprises assaying plasma levels of atherogenic lipoproteins using an assay to obtain plasma levels of assayed atherogenic lipoproteins. Further comprising the step of assaying enzymatically, the assay of plasma esterified cholesterol, the assay of plasma apolipoprotein-B, the gel filtration assay of plasma, the ultracentrifugation assay of plasma, and the precipitation assay having the following components: A plasma ultracentrifugation assay, a sedimentation assay, an immunoturbidimetric assay, an antibody-based assay, an antibody-fragmented assay, and an electrophoretic assay for determining the level of a therapeutically effective amount of each of said liposomes, Here, the component is composed of a polyanion, a divalent cation and an antibody. A method selected from the group consisting of: 25. The angina equivalent state is selected from the group consisting of ischemic wall motion abnormalities, dyspnea, impaired motor tolerance, arrhythmias, reduced cardiac function, and associated pain. Composition. 26. The composition of claim 1, wherein the liposome is suitable for administration to a subject before or during surgery. 27. A kit comprising the composition of claim 1, further comprising an anesthetic or sedative. 28. The composition of claim 1, wherein the liposomes are provided periodically during the treatment period. 29. The administration comprises intravenous bolus administration, intravenous infusion, intravenous administration,
The composition according to claim 1, which is selected from the group consisting of intraperitoneal administration and administration as an adjunct to dialysis. 30. The composition of claim 1, further comprising monitoring cardiac function. 31. The composition of claim 30, wherein the cardiac function is EK.
A composition selected from the group consisting of G abnormality, ST partial change, arrhythmia, evaluation of compartment wall motion, blood viscosity, exercise tolerance and ambulatory EKG monitoring, stress cardiography, cardiography, SPECT and stress SP & CT.