Apolipoproteins as Therapeutic Agents for the Treatment of Vascular and Nonvascular Disease

Evaluation of Lipoproteins/Apolipoproteins as Therapeutic Agents for the Treatment of Vascular and Nonvascular Disease Cesare R. Sirtori, polipoproteins are attractive molecules for potential use in therapeutics, being stable, water-soluA ble, and capable of simultaneously binding both lipids and other molecules. Some apolipoproteins can also interact with cell receptors, regulating both cholesterol biosynthesis and removal. The therapeutic use of apolipoproteins may not necessarily be linked to their lipid-binding properties. Apolipoprotein segments have been proposed for clinical use, and complexes of apolipoproteins with other circulating proteins (e.g. immunoglobulins) may provide a potentially useful tool. Table I shows current or possible uses of apolipoproteins, or whole lipoproteins, in a variety of clinical fields.

INDICATIONS OTHER THAN ATHEROSCLEROSIS

Apolipoprotein A-I: Activation of Sperm Motility: The major sperm-activating capacity in serum is mediated by a fraction with a molecular mass of approximately 250 kilodaltons (kD),1 the sperm-activating protein. This complex contains albumin, apolipoprotein (apo) A-I, and immunoglobulin heavy and light chains.2 At specific concentrations of this protein, sperm motility is increased, as is the adenosine triphosphate (ATP) content of sperm. Although the mechanism by which sperm-activating protein induces activation is unknown, structural studies show that a plausible structure of sperm-activating protein places 1 molecule of apo A-I between the 2 arms of the Fab portion of an immunoglobulin G molecule.3 Immunofluorescence studies in human spermatozoa indicate a direct interaction of sperm-activating protein with the lower part of the sperm head. Use of sperm-activating protein is postulated both for diagnostic and therapeutic purposes in human fertilization. Apo E: Inflammatory Conditions and Gout: Gout, characterized by increased levels of uric acid in plasma, may lead to arthritis, mainly at the lower extremities (toe), with crystal formation and abundant inflow of polymorphonuclear neutrophil leukocytes. Apo E, a basic protein, can bind urate crystals in cultured polymorphonuclear neutrophil leukocytes.4 The effects of apo E might not be only chemical, but also involve lymphocyte maturation, up to the formaFrom the Institute of Pharmacological Sciences, University of Milano, Milan, Italy. Address for reprints: Carlos A. Dujovne, MD, Kansas Foundation for Clinical Pharmacology, 10550 Quivira Road, Suite 240, Overland Park, Kansas 66215, USA.

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©1998 by Excerpta Medica, Inc. All rights reserved.

MD, PhD

tion of active cells. A subfraction of low-density lipoprotein (LDL) with immunoregulatory properties, LDLin, inhibits lymphocyte immortalization, which is also a prerequisite for lymphoid tumor development,5 and apo E, or possibly a modified form of it, has been identified as being responsible for the immunoregulatory properties of LDLin.6 These observations indicate a possible use of apo E for treatment of inflammatory disorders ranging from gouty arthritis to progressive lupus erythematosus and rheumatoid arthritis, up to the control of graft rejection. Protocols for the study of interventions in these acute clinical conditions could follow the guidelines used in studies of joint diseases. Apo E: Neurodegenerative Disorders: Degenerative disorders of the central nervous system may be the result of trauma or infectious disease, or part of a generalized condition. Injured mammalian nerves release a soluble protein of molecular mass 37 kD,7 the secretion of which increases dramatically after injury, accounting for up to 5% of total secretory proteins. This protein, identified as apo E, is produced by the macrophages surrounding the injured nerve. The interest in apo E, particularly apo E polymorphism, in the etiology of Alzheimer’s disease, has also grown considerably. Subjects carrying the E4 isoform of apo E are at enhanced risk of the late-onset form of disease,8 although studies on the mechanism of this putative association have been less than enlightening. The recognition of apo E release from injured nerves has stimulated studies examining the possibility that this may be a mechanism for tissue repair. Released apo E is recognized by high-affinity receptors with characteristics of the B,E receptors,9 which would enhance provision of lipids to growing tissue as a local source for myelin production.10 Cholesterol released from injured nerves becomes associated with apo-E– containing lipoproteins, being available for local reutilization.11 In vitro studies have shown that the E3 isoform may be more effective in inducing nerve regeneration than the E4.12 These observations provide exciting clues to possible management of early b-amyloid accumulation, as occurs in Alzheimer’s disease.13 In in vitro systems, apo E2 and E3 show different effects from E4 on neurite outgrowth and improve neuronal remodeling,14 thus providing a possible tool to decrease b-amyloid accumulation. ApoE might even reverse some of the initial changes in patients presenting with the so-called “age-associated memory impairment.” 0002-9149/98/$19.00 PII S0002-9149(98)00259-8

TABLE I Potential Therapeutic Uses of Apolipoproteins or Lipoproteins Apo/Lipo A-I HDL A-IV E

Atherosclerosis Yes Yes Possibly Yes

Others Activation of sperm motility Endotoxin shock Obesity Gout, inflammatory disorders Neurodegenerative diseases Alzheimer’s disease

Clinical studies should follow the guidelines recently provided for Alzheimer’s disease.15 Apo A-IV: Obesity: A-IV, a structural component of chylomicrons, may play a role in the catabolism of triglyceride-rich lipoproteins and also in reverse cholesterol transport.16 Increased apo A-IV levels in mesenteric lymph may act as a physiologic signal for satiation, and apo A-IV might be an important factor in satiation after fatty meals.17 Decreased feeding, drinking, and ambulation are seen in rats after single A-IV doses of 60 –200 mg, infused via an indwelling right atrial catheter. Apo A-IV dose dependently suppresses food intake by decreasing meal size without changing intervals between meals (speed of eating or latency to the first meal after infusion).18 The effect lasts for 3 hours after the infusion. A central effect of the apolipoprotein was suggested by infusions into the third ventricle instead of intravenous doses. As little as 1 mg/rat infused into the ventricle suppresses food intake by almost 50%, and at 4 mg food intake is almost totally suppressed.19 It is currently being investigated whether apo A-IV by the parenteral route can also decrease food intake in humans. If apo A-IV is found helpful, it will need to be tested in Phase I trials in volunteers with direct evaluation of food intake, and in Phase II trials in obese patients versus established anti-obesity treatments. Apo A-I: Endotoxin Shock: Apolipoproteins, particularly apo A-I, may have an unexpected role in the management of shock due to bacterial endotoxins. The amphipathic helixes of A-I can, in fact, interact with phospholipids on the surface of the endotoxin, in this way potentially decreasing clinical consequences. Whole high-density lipoprotein (HDL) also exerts this effect. Endotoxin shock is a clinical condition resulting from a release of endotoxins from gram-negative bacteria, which is fatal in 30 – 80% of the cases. High plasma HDL concentrations can protect mice against endotoxin in vivo; transgenic animals with 2-fold elevations of plasma HDL show improved survival over low-HDL mice, whereas intravenous infusions of HDL almost totally protect mice from endotoxin shock.20 Animal21 and more recently clinical22 data on the effectiveness of reconstituted HDL have been obtained by mimicking endotoxin shock with intravenous bacterial lipopolysaccharides, generally 4 ng/ kg.23 In healthy volunteers given the lipopolysaccharide challenge, a 4-hour infusion of 40 mg/kg reconstituted HDL, starting 3.5 hours before the chal-

lenge decreases the flu-like symptoms and the release of tumor necrosis factor, interleukin-6, and interleukin-8.23 It also attenuates the changes in leukocyte counts and the enhanced expression of CD11b/CD18 on granulocytes. Clinical pharmacology Phase I studies need to be complemented by controlled Phase II investigations in patients with different types of shock on standard therapy (corticosteroids, antibiotics, parenteral fluids, etc.). In these conditions a blinded study of HDL/A-I can evaluate very well the potential to prevent the most serious sequelae of shock.

ATHEROSCLEROSIS TREATMENT Apolipoproteins or whole lipoproteins may serve as therapeutic tools for arterial plaque removal. Thus 2 or possibly 3 apolipoproteins may be effective in the prevention or reversal of atherosclerosis. Apo E has caused regression of arterial lesions in the Watanabe heritable hyperlipidemic rabbit. The possible beneficial effect of apo A-I is based on studies on atherosclerosis regression induced by HDL or by isolated A-I or (A-IM) infusions. Finally, mice transgenic for apo A-IV show a markedly decreased susceptibility to atherosclerosis. Apo E: The suggested use of apo E for improving lipoprotein metabolism was based on the affinity of apo E-containing lipoproteins for liver cell membranes, responsible for the uptake of dietary cholesterol by way of chylomicron remnants,24 interacting with the chylomicron-remnant receptor.25 Injections of apo E into Watanabe heritable hyperlipidemic rabbits can improve clearance of chylomicron remnants and hence decrease cholesterolemia.26 Infusion of approximately 30 mg of apo E into hypercholesterolemic rabbits decreases plasma cholesterol (8.3% by 1 hour and 19% after 3 hours), and this can be maintained for up to 8 hours. Chronic treatment of Watanabe heritable hyperlipidemic rabbits with thrice-weekly injections of apo E (30 mg per rabbit) for 8.5 months has resulted in a dramatic reduction (240%) of the lesions in these rabbits with genetic hypercholesterolemia.27 Apo A-I/HDL, A-IV: The well-substantiated beneficial effects of elevated HDL levels against arterial lipid deposition suggest that one might test the activity of whole HDL or of apolipoprotein fractions on arterial lipid deposits. The human lipoprotein fraction isolated at density 1.063–1.25 g/mL (very high-density lipoprotein [VHDL]), infused intravenously to cholesterol-fed rabbits (50 mg of protein once a week) throughout the period of cholesterol feeding, markedly decreased sudanophilia and the cholesteryl ester content of the aorta.28 This same fraction actually induced regression of established arterial lesions in the rabbits.29 HDL or A-I exerts a prostacyclin-stabilizing activity30 and apo A-I itself shows a direct fibrinolytic action in in vitro models31 as well as a potent anticytokine activity (see above). Rabbits transgenic for human apo A-I show a markedly lower rate of atherosclerosis development.32 Definitive support of a direct activity of apo A-I in A SYMPOSIUM: CLINICAL TRIAL GUIDELINES

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inhibiting atherosclerosis progression or stimulating regression has come from a number of studies using either apo A-I isolated from plasma33 or recombinant apo A-I obtained with different vectors. Significant data have also come from the use of a recombinant dimer of apo A-IM. A-IM is a natural mutant of apo A-I (cys for arginine at position 173) apparently associated with a decreased risk of arterial disease.34 Kinetic studies have indicated that the dimer of apo A-IM (A-IM/A-IM) has a long plasma half-life35 and shows potent direct fibrinolytic activity, in the presence or absence of tissue plasminogen activator. These observations have prompted evaluations in animal models. In these, a small number of injections of the A-IM dimer, evenly spaced before and after procedures inducing arterial stenosis, resulted in a .50% lower lesion development.36,37 Studies in humans (see also above) have indicated that apo A-I, of extractive or natural source, is very well tolerated, can raise HDL cholesterol in patients with low levels, and does not induce significant antibody formation.38,39 It therefore appears well suited for studies of arterial intervention. Finally, mice transgenic for human apolipoprotein A-IV also show a marked reduction in atherosclerosis susceptibility.40

CLINICAL STUDIES IN ATHEROSCLEROSIS Phase I studies on apolipoproteins should be carried out in healthy volunteers, to detect potential subjective or objective side effects or antibody formation. A dose–response relation should be established by appropriate immunoassays between the amount of recombinant/natural apolipoprotein administered and the rise of plasma levels. Phase II studies can make use of the protein/lipoprotein amounts shown in Phase I to achieve sustained increases of plasma levels. Such studies should be designed to evaluate arterial benefit, assessed for example by ultrasound studies in coronary or peripheral arteries, or by functional studies in different arterial regions such as effort tests, the onset and duration of variant angina and arterial reactivity or dilation after muscarinic stimuli.41 Phase II studies should be designed with specific endpoints to establish that the addition of apolipoprotein to the regimen can definitely improve chances for arterial regression. Studies should be double-blinded, allowing a quantitative assessment of the endpoint versus standard treatments in patients with severe arterial conditions. Tests assessing the safety of treatment (including all parenchymal and hematologic evaluations, in addition to immunologic screening) should allow determination of the safety of the intervention. Phase II studies could be conclusive in themselves if they clearly established therapeutic efficacy of the apolipoprotein. Phase III studies could confirm in large patient series the validity of these novel therapeutic tools. 38F THE AMERICAN JOURNAL OF CARDIOLOGYT

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