THERAPY OF ATHEROSCLEROSIS
This invention relates to the therapy of atherosclerosis.
Atherosclerosis is the arterial disease that gives rise to heart attacks, strokes and poor peripheral circulation. It thickens the walls of arteries, and thereby reduces blood flow through them. It also predisposes to their further narrowing and complete occlusion by inducing the formation of thrombi (blood clots) within them.
An important feature of the atherosclerosis disease is that a type of white cell from the blood, the monocyte, enters the artery wall. Large numbers enter into the localised areas of the disease, named plaques, and there mature into cells called macrophages (1,2). The available evidence suggests that these cells are important in the disease process, and that several further major features of the disease, for example the accumulation of lipid, are secondarily dependent on their presence.
A major route for the entry of the monocytes into the artery wall, particularly in the earlier stages of the disease, is their passage through the thin layer of cells, called the endothelium, which line the interior of the vessel. For this to occur, they have first to adhere to the surface of the cell layer. This adhesion can be observed directly, and there is good evidence that it is produced by the presence of a range of adhesion molecules on the surface, which bind specifically to complementary adhesion molecules on the blood monocytes (3-6). This adherence is similar to that which occurs between white cells and small blood vessels in inflammation, to allow the passage of white cells into the focus of infection or other injury. In both inflammation and atherosclerosis, it is likely that the adhesion is a critical regulatory step governing the rate of cell entry in the vessel wall. As the leukocytes have a central role in driving inflammation, and also most probably in atherosclerosis, it is also likely that the adhesion is also an important regulatory event in the control of the development of atherosclerosis.
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In atherosclerosis, when it reaches an advanced stage, the endothelial lining of cells frequently becomes disrupted, often by a partial cracking of the artery wall. Blood platelets and monocytes then adhere to the damaged region, initiating the formation of a thrombus (blood clot). Platelets are directly involved in the thrombus, and in particular contribute to the formation of this solid mass by binding to fibrinogen or fibrin molecules also present within it via a surface membrane receptor, αIlbβ3 integrin, which is also known in the platelet field as gpIIb/HIa (7). This platelet receptor will also bind to a number of extracellular matrix proteins, including fibronectin and vitronectin. Monocytes are found bound to the internal surface of atherosclerotic arteries, and are also present within thrombi. In both instances they may contribute to the initiation or promotion of the thrombotic process by the production of tissue factor, a molecule that initiates coagulation (8), or by their ability to interact with platelets via the adhesion molecule P-selectin that is present on the platelet surface. The interaction of monocytes with activated endothelial cells, such as are found over atherosclerotic plaques, increases their expression of tissue factor greatly (8). The monocytes can bind to the arterial wall both in areas where the endothelium is intact and activated, and they also bind avidly to areas which have lost their endothelium as a result of damage. Both these types of surface are found with atherosclerosis. Once bound, they may enter the wall, to contribute to the atherosclerotic process.
Studies on the expression of adhesion molecules on the endothelial cells of human atherosclerotic arteries have shown increased levels of the adhesion molecules ICAM- 1, P-selectin and E-selectin specifically in the diseased areas (3-6). In addition, a functional assay was devised to determine the adhesion molecules functionally active in human atherosclerotic tissue, based on a method originally described by Stamper and Woodruff (1976) (6,9,10). In this assay, a suspension of monocytes, or of monocyte-like cell line cells, was added to histological sections of human atherosclerotic tissue mounted on a microscope slide. The slides, covered by a drop of fluid containing the monocytes, were agitated on a stirring platform. Specific adhesion of the monocytes was observed to the atherosclerotic areas of the tissue section, and significantly less to normal areas.
This adhesion was both to the endothelium and to the underlying thickened artery wall. Antibody, peptide and lectin inhibitors of specific adhesion molecules could be added to the assay to determine which diminished the adhesion, and hence which adhesion molecules were active in this system. The results from this work showed that ICAM-1, P-selectin, and the monocyte molecule CD14 were involved (6,9).
The genetically engineered antibody named ReoPro (also known as abciximab) has proved effective at inhibiting restenosis of arteries after surgical unblocking by angioplasty (11), and in the treatment of unstable angina (12). Thus the objects of its therapeutic use are to prevent thrombosis complicating angioplasty, and the thrombosis complicating advanced atherosclerosis associated with unstable angina. This antibody reacts with the αIIbβ3 integrin adhesion molecule on platelets, thus inhibiting platelet adhesion in the formation of thrombi, however it is known that the antibody has some ability to block other integrins, in particular that it also reacts with the αvβ3 integrin, due to the common β3 integrin chain (13). Furthermore, it reacts with the leukocyte integrin Mac-1 ( Mβ2 integrin), and thereby inhibits the binding of leukocytes to fibrinogen and ICAM-1 (13).
The vβ3 integrin is expressed on some monocytes and some endothelial cells, and is well known as a receptor for binding to the extracellular matrix molecule, vitronectin. It also binds onto a number of other proteins, particularly the endothelial adhesion molecule CD31 (22), and the extracellular molecules fibrinogen, fibronectin and collagen (7). It is known to be another adhesion receptor involved in the binding of monocytes to activated endothelium (18), although it is not known to be active with the endothelium of arteries (see discussion below). However studies on arteries have shown that it is expressed on both atherosclerotic and non-affected areas of endothelium. Likewise it was found to be expressed generally in arterial smooth muscle cells (14). Thus no specific association with atherosclerosis has been demonstrated, and no deduction could be reasonably made from this previous work that inhibition of its activity might be beneficial in the therapy of atherosclerosis.
Abciximab has been found to have greater activity in inhibiting restenosis post angioplasty than the more specific α1Ibβ3 integrin inhibitor, integrilin, and it has been suggested that this could be attributed to its more extensive reactivity (15), in particular because of its reactivity with αvβ3 integrin in addition to the platelet αιrbβ3. However no precise indications of the mechanisms by which this reactivity could be of therapeutic advantage have been given.
According to one aspect, the present invention provides the use of an inhibitor of the adhesive functions of αvβ3 integrin for the manufacture of a medicament for use in the therapy of disease caused by atherosclerosis.
According to another aspect, the present invention provides a method for the therapy of disease caused by atherosclerosis which comprises administration of an inhibitor of the adhesive functions of αvβ3 integrin.
The present invention is based on the finding that the αvβ3 integrin has a significant role in the adhesion of monocytes to the artery wall in atherosclerosis and that inhibition of this adhesion by pharmaceutical agents is a means for the therapy of atherosclerosis. As used herein the term "therapy" includes prophylaxis as well as treatment of an existing condition.
According to the present invention, the therapy of atherosclerosis involves inhibiting αvβ3 dependent binding of monocytes to the arterial wall. As already noted, binding of monocytes to the arterial wall is implicated at an early strage in the devcelopment of atherosclerosis (primary atherosclerosis) before the development of complications due to thrombosis. Accordingly, the therapy according to the invention is particularly advantageous in the case of disease caused by atherosclerosis without complications due to thrombosis. For example, the therapy according to the invention can be applied in the case of angina pectoris, cerebral ischemia, peripheral vascular disease or myocardial infarction.
Suitable inhibitors of the adhesive function of the αvβ3 integrin include antibodies against the integrin or other molecules, e.g. small chemical molecules, which inhibit αvβ3 dependent monocyte adhesion.
As far as antibodies are concerned, it is possible to use rodent, for example rat or mouse, monoclonal antibodies against αvβ3. One example of such an antibody is the mouse monoclonal antibody LYP18 (whose production is described in more detail below) or an equivalent monoclonal antibody specific for the same epitope. The monoclonal antibody may be isolated from a hybridoma cell line producing it or may be produced using standard techniques of recombinant DNA technology.
It may also be advantageous to develop anti- vβ3 antibodies which have less potential for eliciting a reaction from the human immune system using known techniques for the production of chimeric or humanised, e.g complementarity determining region (CDR)- grafted, antibodies. Such antibodies may be produced by using recombinant DNA- technology to isolate DNA encoding the rodent monoclonal antibody from the hybridoma cell line producing it and then manipulating the DNA to produce DNA encoding the chimeric or humanised antibody. Thus, DNA encoding the constant region of the rodent antibody may be replaced by DNA encoding a human constant region to produce DNA encoding a chimeric antibody. Alternatively, DNA encoding the CDRs of a human antibody may be replaced by DNA encoding the CDRs of the rodent anti-αvβ3 antibody to produce DNA encoding a CDR-grafted antibody having the same antigen specificity as the rodent antibody. Expression of the DNA encoding the chimeric or humanised antibody in a suitable cell line leads to production of the antibody itself. For example, the above techniques can be used to produce a recombinant antibody, for example a chimeric or CDR-grafted antibody, having at least the same CDRs as the antibody LYP18.
It may be advantageous in a threapeutic context to use small chemical molecules, for example molecules with a molecular weight up to about 1000, which inhibit the adhesive functions of αvβ3 integrin. Some small molecules with this activity are
already known, including for example the compounds disclosed in US-A-5 760 028 (Jadhav et at) and US-A-5 710 159 (Voss et at). Other small chemical molecules having this activity can be identified using an adhesion assay such as that described in reference 9 or the modified version of this assay described in more detail below.
In some cases, it may be possible to administer the inhibitor of the adhesive functions of αvβ3 integrin to a patient as the raw substance but the inhibitor will generally be presented as a pharmaceutical composition. In this context a pharmaceutical composition comprises at least one inhibitor of the adhesive functions of vβ3 integrin (referred to herein as the "active ingredient") with one or more pharmaceutically acceptable carriers or diluents. The carrier(s) or diluent(s) must be "acceptable" in the sense of not having any deleterious effect on the patient and being compatible with other components of the formulation. The pharmaceutical composition may also contain other therapeutic ingredients having the same or a different therapeutic effect from the active ingredient, for example agents having an effect on the heart or circulation, such as anti-coagulants or anti-hypertensives, or inhibitors of other adhesion molecules involved in atherosclerosis such as CD 14.
In the case of small chemical molecules, the active ingredient may be formulated for administration by any suitable means provided that it is delivered to the circulation in such a manner that αvβ3 integrin dependent monocyte adhesion in the vicinity of atherosclerotic plaque or at potential sites of atherosclerotic plaque formation can be inhibited. Examples of suitable forms of administration include oral, parenteral, rectal or intranasal, e.g. by inhalation.
A pharmaceutical composition for oral administration may take the form of, for example, tablets or capsules and may be prepared by processing the active ingredient in a conventional manner together with one or more pharmaceutically acceptable excipients. Tablets may be prepared by compression or moulding in known manner and suitable excipients include binding agents, fillers, lubricants, disintegrants and wetting
agents. Tablets or capsules may be coated in known manner, for example to provide slow or controlled release of the active ingredient.
Liquid preparations for oral administration may take the form, for example, of solutions, syrups or suspensions or may be presented as a dry product for re- constitution with water or another suitable vehicle prior to use.
Compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may be formulated in known manner. The formulations may be presented in unit-dose or multi-dose containers, for example, ampoules or vials, or may be stored in a lyophilised condition suitable for reconstitution by addition of sterile liquid, for example water for injection.
Compositions for rectal administration may be presented in forms such as suppositories or retention enemas which may be formulated in known manner.
Compositions for intranasal administration may be formulated as solutions for administration via a metered dose or unit device or as a powder including a suitable carrier for administration using an appropriate delivery system.
Antibodies which inhibit αvβ3 integrin dependent monocyte adhesion will generally also be administered to patients in the form of a pharmaceutical composition which preferably includes, in addition to the antibody, a physiologically acceptable carrier or diluent, possibly in admixture with one or more other agents such as other antibodies or drugs, such as antibiotics or agents having an effect on the heart or circulation, or inhibitors of other adhesion molecules involved in atherosclerosis such as CD 14. Suitable carriers include physiological saline and phosphate buffered saline. Alternatively the antibody may be lyophilised and reconstituted before use by me addition of an aqueous buffered solution. Routes of administration of the antibody include intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.
The method by which the agent which inhibits αvβ3 integrin dependent adhesion is used in the treatment or prevention of atherosclerosis will depend on the nature of the agent. Small chemical molecules may be used prophylactically over long periods by subjects at risk of atherosclerosis. Antibodies carry more risk of an adverse reaction from the subject's immune system and are more suitable for short term therapy of patients at particular risk in special circumstances, for example following heart transplantation. In all cases the precise dose to be administered will be at the discretion of the attendant physician but will depend on the nature of the agent and a number of other factors including the age and sex of the patient, the condition of the patient and the severity of the disorder being treated.
According to a further aspect, the present invention provides a method of assaying leukocyte binding to a tissue sample which comprises contacting an arterial tissue sample section at a temperature of at least 10°C with a suspension in a suitable medium of adherent cells selected from monocytes and cell lines having adhesion properties to arterial tissue similar to monocytes, and quantitating the number of bound cells over a defined area of tissue sample section characterised in that the adherent cells are biotinylated before addition to the assay, and detected by addition of an enzyme and avidm-containing molecule, and the subsequent development of an enzyme-dependent colour reaction.
Adherent cells may be biotinylated by the action of a solution of a sulpho-NHS-biotin reagent. Detection of the adherent cells may be by the addition of solution containing an avidin-biotin-peroxidase complex, and subsequent addition of a solution containing hydrogen peroxide and diaminobenzidine. Quantitation of the binding of leukocytes may carried out by the use of computer-based colour image analysis. The action of an inhibitor of adhesion may be quantitated by the performance of replicates of the assay with and without the addition of the inhibitor.
The invention is explained further with reference to the following experimental findings and discussion in which reference is made to the accompanying drawings in which:
Figure 1 shows the effect of various antibodies and peptidesd on U937 adhesion; and
Figure 2 shows the effect of various antibodies on monocyte adhesion.
Experimental Ωndings 1. Monocyte Adhesion
The abciximab antibody, and the specific monoclonal antibodies LYP2 and 23C6, reacting with α1Ibβ3 integrins and αvβ3 integrins respectively, were tested in an atherosclerosis adhesion assay. A further antibody LYP18, reacting with the β3 chain in both integrins was also assessed. The mouse myeloma protein MOPC21 was used as a negative control. A CD 14 antibody and a peptide containing the arg-gly-asp sequence were used as positive controls (9). The assay was done using phorbol ester stimulated U937 monocyte-like cells, and isolated peripheral blood monocytes tested on atherosclerotic human carotid artery, as previously described (9), but with the following improvements. The U937 monocyte cells were biotinylated prior to the assay by use of the sulpho-NHS-biotin reagent (Pierce), according to die manufacturer's protocol. After adhesion and fixation, the adherent cells were stained by addition of an avidin-biotin-peroxidase complex (ABC) solution (Dako), made according to the manufacturer's instructions. This was then followed by a diaminobenzidine and hydrogen peroxide solution to give brown staining of the cells. The percentage of the area of the intimal layer of the atherosclerotic arterial wall occupied by the colour reaction was then measured by image analysis, using a Quantimet image analyser (Leica). This measurement gave an index of the extent of monocyte adhesion (20). Multiple triplicate assays were performed with U937 cells and the antibodies, as detailed in Table 1. Two triplicate experiments were then done with peripheral human blood monocytes and the ReoPro antibody, and one with these cells and the αvβ3 integrin antibody 23C6.
TABLE 1
Number of triplicate experiments performed with U937 cells, and with the antibodies and peptide indicated. The mean level of adhesion for each series of experiments in the presence of inhibitors is expressed as a percentage of the control adhesion.
Agents N % inhibition of control
MOPC21 8 11.4
CD14 7 73.56
RGD 4 89.75
ReoPro 12 71.08
(αIIbβ3 + αvβ3)
LYP18 4 89.65
(α1Ibβ3 + vβ3)
23C6 3 87.3
(αvβ3)
n = number of triplicate experiments RGD = arg-gly-asp containing peptide
Abciximab, LYP18, and the specific αvβ3 integrin antibody 23C6 all gave strong inhibition of the binding of U937 cells and blood monocytes to the atherosclerotic artery wall, that was highly statistically significant (Figures 1 and 2).
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Figure 1 shows the effect of MOPC21 mouse immunoglobulin, antibody to CD14, arg-gly-asp peptide, ReoPro, and antibodies LYP18, 23C6 and LYP2 on the adhesion of U937 cells to atherosclerotic human artery. Adhesion is determined as a percentage of the adhesion in the absence of inhibitors as measured in triplicate experiments. The mean ± SD of this percentage is given for the series of experiments on each antibody. The number of experiments done is shown in Table I.
Figure 2 shows the effect of an antibody to CD14, ReoPro, and 23C6 anti αvβ3 integrin on the adhesion of peripheral blood monocytes to human atherosclerotic plaques. Adhesion is determined as in Figure 1.
Adhesion to both the endothelium and the bulk of the arterial wall was inhibited. By contrast, the specific anti-α!Ibβ3 integrin antibody LYP2 gave no significant inhibition of U937 binding, nor did the non-immune mouse immunoglobulin MOPC21.
2. Generation of a monoclonal antibody (LYP18) directed against GPIIb/iπa and αvβ3
Immunisation procedure: Platelets were isolated from blood (50-60 ml), drawn from consenting, healthy, adult donors, and washed by the technique of Mustard & Massini (23). These washed platelets were then treated with chymotrypsin (0.2 mg/ml) for 30 min at 25 °C. Chymotrypsin-treated platelets were then washed once and resuspended in a phosphate buffer saline (PBS) prior to injection in mice. Washing of platelets in the absence of calcium was shown to allow an extensive degradation of the GPUb/IIIa complex and other surface glycoproteins (24).
Screening procedure: Platelets isolated from blood, collected in ACD (anticoagulant), were washed by the technique of Mustard & Massini (23) and treated with chymotrypsin (0.2 mg/ml) for 30 min at.37°C. Platelets used for screening or biological assays were washed with physiological levels of calcium and magnesium. Supernatant of generated hybridomas was selected for their inhibitory effect in
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preventing fibrinogen-induced aggregation of chymotrypsin-treated platelets. One clone (HY109/246 given the name of LYP18 or Lyon Platelet number 18 generated monoclonal antibody) was selected and found to have a very potent effect in inhibiting aggregation of chymotrypsin-treated or normal platelets.
Inhibition of aggregation: The newly identified monoclonal antibody (MoAb) (LYP18) was observed to be a very potent inhibitor of platelet aggregation assayed for its capacity to inhibit platelet aggregation induced by different agonists. MoAb LYP18 (40μg/ml) inhibited the aggregation of chymotrypsin-treated platelets even when using a high concentration of fibrinogen (2mg/ml). LYP18 also inhibited ADP (5μm), collagen (4μg/ml) and thrombin (0.07U/ml)induced-aggregation of control platelets (not treated with chymotrypsin).
LYP18 Binding to platelets: In addition, the biding of 125I-labelled LYP18 to control or chymotrypsin-treated platelets was investigated in the presence or absence of a another monoclonal antibody (LYP2) directed against GPπb/ma. The number of binding sites of LYP18 on chymotrypsin-treated platelets was significantly increased compared to control non-treated platelets. In contrast, LYP2 did not show any binding difference.
Fibrinogen binding to platelets in the presence of LYP18: Binding of 125I-labelled fibrinogen to chymotrypsin-treated platelets was inhibited by 90 % when using LYP18 and by 60% when using LYP2.
LYP18 epitope mapping: Crossed immunoelectrophoresis (CIE) experiments show that 125I-labelled LYP18 bind to the GPHb/HIa complex in the presence of calcium but not to dissociated GPITb or GPIIIa complex in the absence of calcium. LYP18 has been shown to bind to vβ3 expressed by endothelial cells, macrophages, COS cells and melanoma cells (25, 26). Moreover, LYP18 has been shown to compete with c7E3 (Reopro).
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Discussion
The evidence to date does not permit the definite identification of the site of expression of the active αvβ3 integrin responsible for the induction of monocyte binding to the arterial wall. As it is expressed both on activated monocytes, and on cells in the artery wall, either could be responsible, or indeed both. Monocytes and other leukocytes activate on contact with activated endothelium, such as is found over atherosclerotic plaques, through exposure to chemokines such as MCP-1 (16). Integrins change their conformation in an activation process, but at present it is not possible to distinguish activated from non-activated αvβ3 in a tissue section, for instance by reaction with antibody probes. Hence it is only through a functional adhesion assay, including inhibition by a specific antibody, as described here, that its involvement can be definitely recognised.
The monocyte vβ3 receptor has a further biological role that may be relevant. It is involved in the recognition of cells undergoing programmed cell death, apoptosis. Apoptosis occurs in atherosclerotic plaques, in both macrophages and smooth muscle cells, and is induced by oxidised lipoproteins (17).
A distinction has to be made between the anticipated spectrum of activity of an agent such as an inhibitor of αvβ3 integrin, which is likely to have activity principally against the fundamental disease process of atherosclerosis itself, compared to agents which block platelet activity, such as inhibitors of αIIbβ3 integrin. The former have the potential to act at an earlier stage in the disease process, and inhibit its development, so that diseases such as angina pectoris that can depend on the presence of atherosclerosis per se could be inhibited. Furthermore diseases that usually depend on the thrombotic complications of atherosclerosis, such as myocardial infarction, are also less likely to occur through inhibition of the fundamental underlying atherosclerosis. There are further varieties of ischaemic arterial disease in which the dependence on thrombosis is uncertain or variable, for instance peripheral vascular disease, and probably also carotid atherosclerosis. In patients with such diseases,
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therapy with a αvβ3 integrin inhibitor may have greater potential than an anti-platelet agent.
An advantage of a specific anti-αvβ3 integrin agent is likely to be that it would not cause major bleeding complications, as occurs with anti-platelet therapy, including the use of abciximab (21). As such an agent has an ability to block monocyte adhesion to the artery wall, and these cells may contribute to thrombosis, such an agent could have some, probably minor, anti-thrombotic action. Furthermore, αvβ3 integrin contributes to fibrinogen binding directly, thus possibly aiding thrombotic changes around monocytes bearing the molecule. As adherent monocytes are found over atherosclerotic plaques, this action could be valuable in preventing specifically the thrombosis that complicates atherosclerosis.
A number of other adhesion molecules mentioned above have been identified by previous studies as being involved in monocyte-atherosclerotic artery adhesion. Therapeutic regimes could include a combination of agents acting against these in addition to αvβ3 integrin, as it is possible that their actions could summate, or even be synergistic. It is possible that CD 14 might have a role in activating vβ3 integrin (19), and inhibiting both might be effective through acting at two stages on a pathway.
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