Patent Application: US-93426097-A

Abstract:
the present invention relates to methods for treating inflammation in body tissues . more specifically , certain disclosed methods relate to selectively inducing apoptosis in inflammatory immune cells . the methods are particularly suitable for treating inflamed atherosclerotic plaques . the techniques generally involve heating the cells with a catheter that is equipped to produce infrared radiation . the cells are heated for a sufficient time and at a sufficient temperature to induce programmed cell death .

Description:
the following examples are offered by way of illustration and are not intended to limit the invention in any manner . in the examples , all temperatures are in degrees celsius unless otherwise noted . the inventors have made the surprising discovery that macrophages are more susceptible to heat induced apoptosis than endothelial cells . this discovery led to the development of the present techniques that use heat to reduce inflammation in inflamed tissue and especially in inflamed atherosclerotic plaques . however , greater sensitivity to heat - induced apoptosis of macrophages is not a requirement of the invention because many athersclerotic plaques are denuded of endothelium in which case induction of endothelial cell apoptosis would be moot . fig1 shows the percent of cells from watanabe ( atherosclerosis ) rabbit aorta tissue undergoing apoptosis after exposure for 15 minutes at the temperatures shown , followed by &# 34 ; tunel &# 34 ; staining after a 6 hour incubation at 37 ° c . fig2 shows similar results when the cells in human carotid artery endarterectomy samples were investigated in a similar manner . living human carotid atherosclerotic plaque was obtained by endarterectomy , immediately placed in tissue culture and subjected to varying temperatures for fifteen minutes . after four subsequent hours at 37 ° c . these specimens were fixed and processed for light and electron microscopic histology . the sections were subjected to histochemistry for the enzyme terminal deoxynucelotidyl transferase , which results in a blue color in the apoptotic cell . a kit from trevigen , inc ( gaithersburg , md ., 20877 ) was used with the appropriate positive and negative controls . as a further control the stained cells were evaluated by electron microscopy . the results show a significant number of apoptotic cells at 37 ° c . there was an increase in the number of apoptotic cells at 42 ° c . with a peak at 45 ° c . these results were confirmed by electron microscopic finding of chromatin margination , protrusions and nuclear fragmentation and budding with the production of membrane bound apoptotic bodies . light microscopy suggested that almost all of the cells undergoing apoptosis were macrophages . finally , a minority of endothelial cells underwent apoptosis with 15 minutes of exposure to 45 ° c ., suggesting that there is indeed a window in which the macrophages can selectively be induced to undergo apoptosis . fig3 shows more directly the relationship between cell density and heat produced as measured by an infrared camera . the elevated heat produced in areas having elevated numbers of macrophages facilitates a detection method for identifying inflamed plaques as discussed in u . s . patent application . ser . no . 08 / 717 , 449 . in fig4 is shown the results from a study in which a representative specimen of human carotid endarterectomy tissue was divided and half incubated at 44 ° c . for 15 minutes in a humidified incubator , followed by 6 hours at 37 ° c . &# 34 ; tunel &# 34 ; staining for dna was employed to demonstrate apoptosis . these cells are indicated by the dark stain . no counterstain was used in this section . the capillary nuclei are faintly shown as unstained macrophage nuclei . the photograph demonstrates the difference between the nuclei of heated and unheated cells . typically , heat induced apoptosis of inflammatory cells to prevent rupture and / or thrombosis of atherosclerotic plaques in the coronary , carotid , iliac femoral or superficial femoral arteries will be carried out in patients presenting with symptoms of ischemia . for example , patients with angina or a positive stress test , or patients with a recent myocardial infarction who are undergoing coronary angiography , will have an infrared catheter passed down the artery in a fashion similar to that of intravascular ultrasound or angioscopy , as described further below . some patients will be referred for this procedure for other reasons . for example , patients having plasma that shows biochemical evidence of inflammation or thrombosis , or endothelial damage or silent myocardial damage , may require coronary catheterization . alternative tests which might bring non - symptomatic people to coronary angiography and infrared testing might include a magnetic resonance imaging scan which can give a kind of non - invasive thermometry , or positron emission tomography , which gives a non - invasive image of glucose utilization ( this may indicate macrophage presence because of their high glucose consumption when activated ). the patients who come for peripheral angiography come either because of claudication or embolism to the feet or because a doctor has found diminished pulses on physical examination . patients requiring carotid angiography typically have had a stroke or transient ischemic attack or a bruit has been detected on physical examination or a carotid narrowing has been detected in the course of magnetic resonance imaging , doppler imaging or angiography performed for other reasons . in the patients described above , following routine angiography , a heat detecting probe , such as is described in u . s . patent application ser . no . 08 / 717 , 449 , will be used to identify lesions that are significantly hotter than the rest of the artery . lesions at higher risk of rupture are two degrees warmer than adjacent tissue . these lesions could be detected by an imaging catheter consisting of any of several fibers that conduct heat , bundled into a standard coronary or other angiographic catheter ranging from four french to seven french in diameter . alternatively , a catheter with standard electrodes on its surface could be used . in one method this will be a balloon catheter made of a compliant ( soft ) balloon material , so as not to damage the endothelium or disrupt the plaque itself . additional evidence that a particular lesion may pose a high risk to the patient , even though the stenosis may be no more than twenty or thirty percent in cross sectional diameter , may be provided by other techniques such as intravascular ultrasound ( to determine how thin the fibrous cap is ) optical coherence tomography which detects cracks in the plaque surface , and / or angioscopy which detects superficial thrombosis . treatment of an inflamed lesion will be performed in several ways . one method is to gently heat the inflamed tissue with heat from about 38 . 5 ° c . to approximately 44 ° c . the treatment is gentle so as not cause cell death due to protein denaturation , desiccation , vesication and / or necrosis . this heating step will be carried out for approximately 15 minutes . this treatment will trigger programmed cell death ( apoptosis ) in the inflammatory cells and spares endothelial cells . a catheter equipped to radiate heat will be used in this method by placing the tip at the location of the &# 34 ; hot &# 34 ; plaque and directing heat into the plaque . subsequently , thermography can be repeated to determine the success of the treatment . lower amounts of heat will also be used to treat inflamed tissue . for example , heating to 38 . 5 ° to 41 ° ( which induces apoptosis in a smaller percentage of macrophages ) will have the beneficial effect of decreasing inflammation produced by macrophages by reducing the production of cytokines such as interleukin 1 and interferon gamma . likewise heating times can be varied . thus , it is envisioned that some treatments will be for approximately 60 minutes , particularly when lower temperatures will be used . in some cases heating times may be as short as 5 minutes , especially when higher temperatures will be used . adjunctive treatments will include the use of cytokines that are known to deactivate macrophages . examples of cytokines envisioned for these treatments include tgf - b1 and tnf - α . other adjunctive therapies will be those directed to preventing the attachment of cytokines to their receptors , the attachment of monocytes , lymphocytes or neutrophils to cells , the expression of selectins or cytokines or chemotactic factors by endothelial cells , soluble receptors and other antagonists of inflammatory cytokines and chemotactic factors , as well as chemical treatments that destroy inflammatory cells . the present invention has been described in terms of particular embodiments found or proposed to comprise preferred modes for the practice of the invention . it will be appreciated by those of skill in the art that , in light of the present disclosure , numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention . for example , a catheter equipped with a laser or other heat source can be substituted for a catheter that produces infrared radiation . in addition , this technique could be adapted to prevent or delay the onset of tissue rejection and treatments of other inflamed tissues , such as restenosis after balloon angioplasty or related interventions including stenting and rotational or directional atherectomy ( since macrophage density in these tissues predicts restenosis ( moreno ) elimination of macrophages by heat - induction of apoptosis will reduce the likelihood that restenosis will occur ). another application will be stenosis of arteriovenous fistulae , dialysis grafts , and other vascular prostheses . in these applications , heat therapy can be applied either from within the vessel or across the skin by means of infrared radiation , radiofrequency , heated metal , etc . still another application would be the use of microwave or radiofrequency to preferentially heat a metal stent to induce macrophage apoptosis to prevent stenosis or resterosis . all such modifications are intended to be included within the scope of the appended claims . the following references to the extent that they provide procedural details supplementary to those set forth herein , are specifically incorporated herein by reference . belli j . a . and bonte f . j . influence of temperature on the radiation response of mammalian cells in tissue culture . radiat . res . 1963 ; 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