USOO6319682B1 (12) United States Patent (10) Patent No.: US 6,319,682 B1 Hochman (45) Date of Patent: *Nov. 20, 2001

(54) METHODS AND SYSTEMS FOR ASSESSING (51) Int. Cl." ...... C12O 1/02; C12O 1/00; BIOLOGICAL MATERIALS USING OPTICAL C12O 1/24; C12O 1/18 AND SPECTROSCOPIC DETECTION (52) U.S. Cl...... 435/29; 435/4; 435/30; TECHNIQUES 435/288.7; 435/32 (58) Field of Search ...... 435/29, 4, 30, (75) Inventor: Daryl W. Hochman, Seattle, WA (US) 435/288.7, 32 (73) Assignee: Cytoscan Sciences, L.L.C., Seattle, WA (56) References Cited US (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,585,401 12/1996 Bandt et al...... 514/562 patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS 2000037616 12/1999 (WO) ...... 435/29 This patent is Subject to a terminal dis claimer. * cited by examiner Primary Examiner Louise N. Leary (21) Appl. No.: 09/629,046 (74) Attorney, Agent, or Firm-Ann W. Speckman (22) Filed: Jul. 31, 2000 (57) ABSTRACT Related U.S. Application Data Optical detection techniques for the assessment of the physi ological State, health and/or viability of biological materials (63) Continuation-in-part of application No. 09/326,008, filed on are provided. Biological materials which may be examined Jun. 4, 1999, now Pat. No. 6,096.510, which is a continu- using Such techniques include cells, tissues, organs and ation-in-part of application No. 08/949,416, filed on Oct. 14, Subcellular components. The inventive techniques may be 1997, now Pat. No. 5,976,825, which is a continuation of emploved in high throughput Screening of potential diag application No. 08/539,296, filed on Oct. 4, 1995, now Pat. ploy 9. gnp 9. p 9. No. 5,902,732. nostic and/or therapeutic agents. (60) Provisional application No. 60/088,494, filed on Jun. 8, 1998. 53 Claims, 11 Drawing Sheets

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US 6,319,682 B1 1 2 METHODS AND SYSTEMS FOR ASSESSING functions, the time course of the physiological effect, gen BIOLOGICAL MATERIALS USING OPTICAL eral cell or tissue health, or the like. AND SPECTROSCOPIC DETECTION Optical techniques have been developed and used for TECHNIQUES Several applications. Light Scattering has been used in the past to provide measurements of OSmotic water permeability REFERENCE TO RELATED APPLICATIONS in Suspensions of osmotically responsive vesicles and Small This application is a continuation-in-part application of cells. A. S. Verkman, “Optical Methods to Measure Mem U.S. patent application Ser. No. 09/326,008, filed Jun. 4, brane Transport Processes,” J. Membrane Biol. 148:99-110, 1999, now U.S. Pat. No. 6,096,510, issued Aug. 1, 2000, 1995. Another study reported a method for the optical which is a continuation-in-part of U.S. patent application measurement of OSmotic water transport in cultured cells. M. Ser. No. 08/949,416, filed Oct. 14, 1997, now U.S. Pat. No. Echevairia, A. S. Verkman, “Optical Measurement of 5,976,825, issued Nov. 2, 1999, which is a continuation of Osmotic Water Transport in Cultured Cells: Role of Glucose U.S. patent application Ser. No. 08/539,296, filed Oct. 4, Transporters,” J. Gen. Physiol. 99:573–589, 1992. 1995, now U.S. Pat. No. 5,902,732, issued May 11, 1999. Optical techniques for observing nerve activity and neu This application claims benefit of Ser. No. 60/088,494, filed 15 ronal tissue are well-established. Hill and Keynes observed Jun. 8, 1998. that the nerve from the waking leg of the Shore crab normally has a whitish opacity caused by light Scattering, FIELD OF THE INVENTION and that opacity changes evoked by electrical Stimulation of that nerve were measurable. Hill, D. K. and Keynes, R. D., The methods and Systems of the present invention employ “Opacity Changes in Stimulated Nerve,” J. Physiol. optical, or spectroscopic, detection techniques for assessing 108:278-281, 1949. Since the publication of those results, the health, physiological condition, and viability of biologi experiments designed to learn more about the physiological cal materials. Such as tissues, cells, and Subcellular mechanisms underlying the correlation between optical and components, and may be used in both in vitro and in vivo electrical properties of neuronal tissue and to develop Systems. One important application of the methods and 25 improved techniques for detecting and recording activity apparatus of the present invention is high throughput Screen evoked optical changes have been ongoing. ing of candidate agents and conditions to evaluate their Suitability as diagnostic or therapeutic agents. Intrinsic changes in optical properties of cortical tissue have been assessed by reflection measurements of tissue in response to electrical or metabolic activity. Grinvald, A., et BACKGROUND OF THE INVENTION al., “Functional Architecture of Cortex Revealed by Optical Drug development programs rely on in vitro Screening Imaging of Intrinsic Signals,” Nature 324:361-364, 1986; assays and Subsequent testing in appropriate animal models Grinvald, et al., “Optical Imaging of Neuronal Activity, to evaluate drug candidates prior to conducting clinical trials Physiological Reviews, Vol. 68, No. 4, October 1988. Grin using human Subjects. Screening methods currently used are Vald and his colleagues reported that Some slow Signals from generally difficult to Scale up to provide the high throughput 35 hippocampal Slices could be imaged using a CCD camera Screening necessary to test the numerous candidate com without Signal averaging. pounds generated by traditional and computational means. A CCD camera was used to detect intrinsic Signals in a Moreover, Studies involving cell culture Systems and animal monkey model. Ts'o, D. Y., et al., “Functional Organization model responses frequently don’t accurately predict the 40 of Primate Visual Cortex Revealed by High Resolution responses and Side effects observed during human clinical Optical Imaging,” Science 249:417-420, 1990. The tech trials. nique employed by Ts'o et al. Would not be practical for Conventional methods for assessing the effects of various human clinical use, Since imaging of intrinsic Signals was agents or physiological activities on biological materials, in achieved by implanting a stainleSS Steel optical chamber in both in vitro and in Vivo Systems, generally are not highly 45 the skull of a monkey and contacting the cortical tissue with Sensitive or informative. For example, assessment of the an optical oil. Furthermore, in order to achieve Sufficient effect of a physiological agent, Such as a drug, on a popu Signal to noise ratios, Ts'o, et al., had to average images over lation of cells or tissue grown in culture, conventionally periods of time greater than 30 minutes per image. provides information relating to the effect of the agent on the The mechanisms responsible for intrinsic Signals are not cell or tissue population only at Specified points in time. 50 well understood. Possible Sources of intrinsic Signals include Additionally, current assessment techniques generally pro dilation of small blood vessels, changes in blood flow, vide information relating to a single or a Small number of Volume and oxygenation, neuronal activity-dependent parameters. Candidate agents are Systematically tested for release of potassium, and Swelling of neurons and/or glial cytotoxicity, which may be determined as a function of cells caused, for example, by ion fluxes or osmotic activity. concentration. A population of cells is treated and, at one or 55 Light having a wavelength in the range of 300 to 3000 nm Several time points following treatment, cell Survival is may also be reflected differently between active and quies measured. Cytotoxicity assays generally do not provide any cent tissue due to increased blood flow into regions of higher information relating to the cause(s) or time course of cell neuronal activity. Yet another factor which may contribute to death. intrinsic signals is a change in the ratio of oxyhemoglobin Similarly, agents are frequently evaluated based on their 60 and deoxyhemoglobin in blood. physiological effects, for example, on a particular metabolic U.S. Pat. No. 5,215,095 discloses methods and apparatus function or metabolite. An agent is administered to a popu for real time imaging of functional activity in cortical areas lation of cells or a tissue Sample, and the metabolic function of a mammalian brain using intrinsic Signals. A cortical area or metabolite of interest is assayed to assess the effect of the is illuminated, light reflected from the cortical area is agent. This type of assay provides useful information, but it 65 detected, and digitized images of detected light are acquired does not provide information relating to the mechanism of and analyzed by Subtractively combining at least two image action, the effect on other metabolites or metabolic frames to provide a difference image. Allowed U.S. patent US 6,319,682 B1 3 4 application Ser. No. 08/474,754 discloses similar optical processes, Volume-regulatory responses, gene expression, methods and apparatus for optical detection of neuronal endocytosis, pinocytosis, ion homeostasis, immune tissue and activity. responses, neurological activity and inhibition, responses to mechanical trauma, chemical insult, and the like. Various U.S. Pat. No. 5,438,989 discloses a method for imaging events, including disease States, dysfunction, inflammation, margins, grade and dimensions of Solid tumor tissue by exposure to pathogens, pollutants, radiation, , illuminating the area of interest with high intensity electro infectious or other agents, aging, apoptosis, necrosis, magnetic radiation containing a wavelength absorbed by a oncogenesis, genetic modification, and the like, affect one or contrast agent, obtaining a background Video image of the more of these physiological processes, producing measur area of interest, administering a contrast agent, and obtain able and predictable changes in the characteristic geometri ing Subsequent Video images that, when compared with the cal Structure or optical properties of individual cells and/or background image, identify the Solid tumor tissue as an area cell populations. of changed absorption. U.S. Pat. No. 5,699,798 discloses The methods and Systems of the present invention employ methods and apparatus for optically distinguishing between optical, or spectroscopic, detection techniques to assess the tumor and non-tumor tissue, and imaging margins and physiological State of biological materials including cells, dimensions of tumors during Surgical or diagnostic proce 15 tissues, organs, Subcellular components and intact organisms. dures. The biological materials may be of human, animal, or plant U.S. Pat. No. 5,465,718 discloses a method for imaging origin, or they may be derived from any Such materials. tumor tissue adjacent to nerve tissue to aid in Selective Static and dynamic changes in the geometrical Structure resection of tumor tissue using Stimulation of a nerve with and/or intrinsic optical properties of the biological materials an appropriate paradigm activate the nerve, permitting imag in response to the administration of a physiological chal ing of the active nerve. The 718 patent also discloses lenge or a test agent, are indicative and predictive of changes methods for imaging of cortical functional areas and dyS in the physiological State or health of the biological material. functional areas, methods for visualizing intrinsic Signals, Two different classes of dynamic phenomena are and methods for enhancing the Sensitivity and contrast of observed in viable biological materials using optical detec images. U.S. Pat. No. 5,845,639 discloses optical imaging 25 tion techniques: (1) geometrical changes in the diameter, methods and apparatus for detecting differences in blood Volume, conformation, intracellular space of individual cells flow rates and flow changes, as well as cortical areas of or extracellular space Surrounding individual cells, and (2) neuronal inhibition. changes in one or more intrinsic optical properties of indi U.S. Pat. Nos. 5,902,732 and 5,976,825 disclose methods vidual cells or of cell populations, Such as light Scattering, for Screening drug candidate compounds for anti-epileptic reflection, absorption, refraction, diffraction, birefringence, activity using glial cells in culture by OSomotically Shocking refractive index, Kerr effect, and the like. Both classes of glial cells, introducing a drug candidate, and assessing phenomena may be observed Statically or dynamically, with whether the drug candidate is capable of abating changes in or without the aid of a contrast enhancing agent. Geometri glial cell Swelling. These patents also disclose a method for cal changes may be assessed directly by measuring (or Screening drug candidate compounds for activity to prevent 35 approximating) the geometrical properties of individual or treat symptoms of Alzheimer's disease, or to prevent CNS cells, or indirectly by observing changes in the optical damage resulting from ischemia, by adding a Sensitization properties of cells. Changes in optical properties of indi agent capable of inducing apoptosis and an osmotic stressing vidual cells or cell populations may be assessed directly agent to CNS cells, adding the drug candidate, and assessing 40 using Systems of the present invention. whether the drug candidate is capable of abating cell Swell Observation and interpretation of geometrical and/or ing. A method for determining the viability and health of intrinsic optical properties of individual cells or cell popu living cells inside polymeric tissue implants is also lations is achieved in both in vitro and in vivo systems disclosed, involving measuring dimensions of living cells without altering characteristics of the Sample by applying inside the polymeric matrix, OSmotically shocking the cells, 45 physiologically invasive materials, Such as fixatives. Physi and then assessing changes in cell Swelling. ASSessment of ologically non-invasive contrast enhancing agents, Such as cell Swelling activity is achieved by measuring intrinsic Vital dyes, may be used in desired applications to enhance optical signals using an optical imaging Screening apparatus. the Sensitivity of optical detection techniques. In applica SUMMARY OF THE INVENTION tions employing contrast enhancing agents, the optical 50 detection techniques are used to assess extrinsic optical Cells from nearly every organ and tissue, of both plant and properties of the biological materials. animal origin, can be dissociated into Single cells, grown and Detection and analysis of the geometrical and/or intrinsic propagated using cell culture techniques. Pathological cells optical properties of individual cells or Sample cell popula from diseased or dysfunctional tissue can also be isolated tions provides information permitting the classification of and maintained in tissue culture. Slices of tissue or tumors 55 the physiological State of individual cells or Sample cell may be maintained under culture conditions for prolonged populations. Based on analysis of the geometrical and/or periods of time and assessed according to methods of the optical properties of a Sample cell population, the Sample present invention. Short-term experiments may also be may be classified as viable or non-viable, apoptotic, conducted on living acute tissue Slices that are prepared and necrotic, proliferating, in a State of activity, inhibition, maintained under physiological conditions. Many multicel 60 Synchronization, or the like, or in any of a variety of lular Systems may also be maintained as functioning Systems physiological States, all of which produce distinct geometri in cell culture. Healthy, pathogenic and dysfunctional cells cal and/or optical profiles. The methods and Systems of the and tissue may also be tested and observed in Situ in animal present invention therefore provide for identification of the models. physiological State of a Sample population and differentia All cells undergo physiological processes that contribute 65 tion among various physiological States. to and determine their geometrical Structure and optical An important application of the methods and Systems of properties. These physiological processes include metabolic the present invention involves Screening cell populations to US 6,319,682 B1 S 6 assess the effect(s) of exposure to various types of test agents apparatus of the present invention with reference to in vitro or test conditions, including candidate compounds and cell populations, wherein intrinsic optical properties of an combinations, drugs, hormones and other biological agents, acute rat hippocampal Slice maintained in a Submerged toxins, infectious agents, physiological Stimuli, radiation, perfusion chamber are monitored at intervals during a con chemotherapy, and the like. The effect of various test agents trol period and an activation period, and data is processed and conditions may be evaluated on both normal and patho logical Sample populations. Safety and cytotoxicity testing is according to methods of the present invention. conducted by exposing a Sample population to a test agent FIGS. 2A-2C show the effect of the agent furosemide on or test condition and assessing the physiological State of the Stimulation-evoked afterdischarge activity in a hippocampal Sample population using optical techniques at one or more Slice comparing the field response measurements at an time points following administration of the test agent or test extracellular electrode, with images highlighting changes in condition. Such testing may be conducted on various Sample optical properties. Experiments were conducted as described populations to determine how a test agent or condition in Example 1. affects a desired target Sample population, as well as to FIG. 3A illustrates an enlarged grey-Scale image of an predict whether a test agent or condition produces physi acute rat hippocampal tissue slice, and ological Side effects on Sample populations that are not the 15 FIGS. 3B-3E illustrate enlarged, contrast-enchanced target of the test agent or condition. images acquired as described in Example 1. According to a preferred embodiment, a disease State or compromised condition is simulated in biological materials FIG. 4A illustrates a view of human cortex just anterior to prior to administration of a test agent or test condition to face-motor cortex with one recording (R) and two stimulat assess the Suitability of the test agent or condition for ing (S) electrodes, and four sites (labeled 1, 2, 3, and 4), treating the disease State or compromised condition. Expo where average percent changes in corresponding optical Sure of Sample populations to a physiological challenge, properties were determined as described in Example 2. Such as a change in extracellular osmolarity or ion FIGS. 4B-4D illustrate plots of the percent optical concentration, altered oxygen or nutrient or metabolite changes in absorption in various spatial regions shown in conditions, drugs or diagnostic or therapeutic agents, a 25 FIG. 4A during electrical stimulation of the human cortex. disturbance in ion homeostasis, electrical Stimulation, Experiments were conducted as described in Example 2. inflammation, infection with various agents, radiation, and FIGS. 5A2-5C4 illustrate spatial images of stimulation the like, Simulates a disease State at a cellular or tissue level. induced epileptiform activity. The images show compari Subsequent exposure of the Sample populations a test agent Sons between different degrees of activation illustrating both or condition and detection and analysis of changes in the Spatial extent and amplitude of optical changes indica geometrical and/or optical properties of the Sample popula tive of the extent of cortical activity. Experiments were tions provides information relating to the physiological State conducted as described in Example 2. of the sample populations produced by the test agent or FIGS. 6A-6H illustrate percentage difference images in condition. Screening techniques may be adapted for use with which the magnitude of optical change indicates the regions various types of cell Sample populations maintained in vitro 35 of greater cortical activity. Experiments were conducted as under appropriate cell culture conditions to provide a high throughput, automated Screening System. Alternatively, described in Example 2. Screening techniques may be adapted to examine cell and FIGS. 7A-7H illustrate percentage difference images tissue populations using various animal models to assess the representing a real time Sequence of dynamic changes of effect of a physiological challenge and/or administration of 40 electrical Stimulation-evoked optical changes in human cor a test agent on various cell populations in animal models in tex. Experiments were conducted as described in Example 2. situ. Screening techniques of the present invention may also FIGS. 8A1-8B3 illustrate functional mapping of human be implemented to examine cell and tissue populations, language (Broca's area) and tongue and palate Sensory area using animal and plant models, to assess the effect(s) of in an awake human patient as described in Example 3. genetic modifications of Such animal and plant models in 45 FIGS. 8A1 and 8B2 illustrate control percentage differ situ. ence images and Changes in geometrical and/of optical properties of indi FIGS. 8A3 and 8B3 illustrate peak optical change images vidual cells or cell populations may be determined by following cortical Stimulation. reference to empirically determined Standards for Specific FIGS. 9A and 9B show time course and magnitude plots cell types, cell densities and various physiological States, or 50 of dynamic optical changes in human cortex evoked in appropriate controls may be run in tandem with the test tongue and palate Sensory areas and in Broca's area Samples to provide direct comparative data. Data is collected (language). Experiments were conducted as described in and, preferably, Stored at multiple time points to provide data Example 3. relating to the time course of the effect of a test agent or condition on Sample populations. Strategies for designing 55 FIGS. 10A-10D illustrate the cranial Surface of a rat, Screening protocols, including appropriate controls, multiple imaged through the intact cranium, and using a contrast Samples for Screening various dosages, activities, and the enhancing agent to highlight areas of optical change. Experi like, are well known in the art and may be adapted for use ments were conducted as described in Example 4. with the methods and Systems of the present invention. DESCRIPTION OF PREFERRED 60 DESCRIPTION OF THE FIGURES EMBODIMENTS Preferred embodiments of the methods and systems for The following description of preferred embodiments assessing biological materials using optical detection tech includes detailed descriptions of Specific applications, as niques of the present invention will be described with well as Specific methods and apparatus. These specific reference to the figures, in which: 65 embodiments are described for purposes of illustrating the FIG. 1 shows a partially schematic flow diagram illus Scope of the invention; the invention is not limited to these trating exemplary methods and output of the methods and applications. Techniques for acquiring data relating to opti US 6,319,682 B1 7 8 cal properties of various types of tissues that would be (ALS) and other types of neuronal injury inflicted by suitable for use with the methods and systems of the present ischemia, hypoglycemia or excitotoxic agents. Trauma, invention are described in numerous U.S. Patents. U.S. Pat. Stroke, excitoxicity and hypoxia are conditions of ischemia Nos. 5,215,095, 5,438,989, 5,699,798, 5,465,718, 5,845, 639, 5,902,732 and 5,976,825 are hereby incorporated that are associated with extensive neuronal cell death in herein by reference in their entireties. Sensitive brain regions. Recent Studies demonstrated that, One important application of methods and Systems of the after experimentally induced ischemia, characteristic apop present invention is to classify the physiological condition or totic DNA fragmentation occurs in affected brain regions. State of biological materials based on their geometrical Methods and Systems of the present invention for classifying and/or optical properties, and to distinguish among various cell populations as apoptotic, necrotic, viable, non-viable, physiological conditions. Viable cell populations are distin and the like, are useful for identifying the physiological State guishable from non-viable cell populations in both in vitro of cells in both in vitro and in vivo systems, as well as for cell Sample populations, and in Situ in animal models based Screening various test agents to ascertain whether they are on a comparison of geometrical and/or optical properties. useful as diagnostic or therapeutic agents. Similarly, Sample populations that are proliferating, or that 15 are responding to various Stimuli by mounting certain Methods and Systems of the present invention may also be responses, Such as immune responses, inflammatory used to identify physiological conditions associated with responses, and the like, are distinguishable from non and to evaluate test agents and conditions for diagnosis and responsive Sample populations. treatment of various disorders, and pathological conditions, Cells or cell populations undergoing apoptosis, an active including migraine headaches, spreading depression, programmed cell death phenomenon, are likewise distin epilepsy, Alzheimer's disease, multiple Sclerosis, psychiatric guishable from cells or cell populations undergoing necro disorderS Such as depression, anxiety, bipolar disorder, Sis. Necrosis may result from mechanical injury, exposure to Schizophrenia, Parkinson's disease and other neurodegen toxins, anoxia due to impairment of the blood Supply, or the erative disorders, inflammation, trauma, malignancies Such like. The physiological changes observed during necrosis 25 as , angiogenesis, Wound healing, immune include Swelling, clumping of chromatin, and deterioration deficiencies, and the like. Test agents and conditions may of the organelles, followed by lysis with release of the cell contents, which are then phagocytized by macrophages. The also be tested for Safety and efficacy for applications Such as cytological changes associated with apoptosis are very dif toxicology, learning and memory, bone growth and ferent and include an early condensation of chromatin and maintenance, muscle and blood Systems, Sensory-input degradation of DNA, and cell volume decreases, with the Systems, and the like. cell membrane and the organelles remaining intact. Apop Optical contrast enhancing agents useful for enhancing totic cells ultimately fragment into several membrane the Sensitivity of optical detection for various types of cells, bounded globules that are phagocytized by neighboring physiological States, and the like, may also be Screened and cells. Neutrophils and macrophages are not involved in the 35 identified using methods and Systems of the present inven terminal Stages of apoptotic processes to the same extent that tion. Sample populations comprising malignant, they are in necrotic processes. pathological, or dysfunctional cells may be exposed to test Identifying cell populations undergoing apoptosis is agents, for example, to identify agents that preferentially important for numerous reasons. Certain genes responsible identify and distinguish malignant, pathological, and dyS for regulating apoptosis play a role in cancer, and cancer 40 therapy by irradiation, chemotherapy, and hormone treat functional cells or tissue. ment all induce apoptosis in tumor cells. Some may, According to one embodiment, methods and Systems of in fact, result from the down-regulation of genes that nor the present invention acquire and compare data representa mally cause apoptosis. Hence, methods for Screening cell tive of one or more dimensional properties of individual populations to ascertain whether or not they are apoptotic is 45 cells or cell Samples. Acquisition, processing and analysis of central to gaining an insight into various pathological data relating to optical properties is described throughout conditions, Such as cancer. Since different agents used in this description. Acquisition of data relating to dimensional cancer treatment induce apoptosis, it is likely that apoptotic properties of individual cells is described below. Acquisition pathways are indicative of the outcome of chemotherapy. and analysis of data relating to dimensional properties may There are many other instances of apoptosis during both 50 be achieved using the same or Similar methods and appara normal and pathological cellular activities. For example, tus described herein with reference to optical properties. hormones regulate apoptosis in gonadal tissues So that numbers and development of Sperm and egg cells are In Sparsely populated cell Samples, cell areas may be regulated. In the immune System, apoptosis plays a clear role approximated using a single plane of focus. If it is desired in the Selection of lymphocytes. Immunodeficiency may be 55 to calculate Volume, the Z-axis (focus) can be automatically caused by lymphocyte developmental blocks which lead to adjusted as well. For example, as an automated and con apoptosis by default. Cell death in these cases is the normal, trolled Stage moves, the optically transparent container con programmed response in the absence of an essential Survival taining the Sample population is positioned So that a Series Signal. However, active induction of programmed cell death of data Sets for multiple, Spatially resolved areas of interest can also elicit immunodeficiency, as in acquired immuno 60 can be acquired, each image being acquired at a predeter deficiency syndrome, AIDS. Considerable evidence Sup mined focal plane. The Volume for each Z-plane can be ports the proposition that HIV activates T cell apoptosis. In approximated (see algorithm below) and then the Volumes the nervous System, apoptosis plays not only a pivotal role during embryogenesis, but also occurs in the adult, generally for each Z-coordinate added together. under various pathological conditions that are accompanied 65 General techniques for approximating cell areas and by devastating consequences for the patients. Examples Volumes, based on Doughty, S., "Calculating property for include Alzheimer's disease, amyotrophoic lateral Sclerosis solids of revolution,” Machine Design, pp 184-186, Dec. US 6,319,682 B1 9 10 10, 1981, are described below. These techniques are based areas of interest in cell Sample populations. Changes in on Green's theorem: optical properties that are indicative of physiological activity and that may be detected include, for example, reflection, 8 O 3P refraction, diffraction, absorption, Scattering, birefringence, Pdx+ody) II (E-Caxdy refractive index, Kerr effect, and the like. Changes in optical (boundary) (area) properties are detected directly using photon Sensitive ele ments and, optionally, optical elements that enhance the detected optical properties. Individual cells are examined using an appropriate mag High resolution detection of dynamic geometrical and nifying device. Edge detection of cell boundaries is achieved optical properties indicative of physiological activity may be using, for example, a Sobel operator. The boundary is accomplished without using dyes or other types of contrast approximated by fitting it to a plurality of Straight line enhancing agents according to the methods and apparatus of Segments of “n” line Segments by “n” nodes. The integration the present invention, as evidenced by the examples of the boundary may be taken as “n” line integrals as described herein. Many of the assessment techniques and follows: 15 apparatus of the present invention are physiologically noninvasive, in that detection and analysis of geometrical X3,3 X,Y and/or intrinsic optical information does not require direct .) as contact of the area of interest with any agents Such as dyes, (.ds + X2, Y2 (.ds +...+ X (.. oils, devices, or the like. For particular applications, it may, however, be useful to administer contrast enhancing agents There are three cases: that amplify differences in an optical property being detected as a function of physiological activity prior to acquiring Case 1: a vertical line, X=constant; Subsequent data and generating a comparison. The use of Case 2: a horizontal line, y=constant; contrast enhancing agents is described in detail, with refer Case 3: an inclined line, y=5(x-x)+y, 25 ence to optical imaging of tumor and non-tumor tissue, in The area is thus: U.S. Pat. No. 5,465,718 and U.S. Pat. No. 5,438,989, which are incorporated by reference herein in their entireties. Suitable contrast enhancing agents include fluorescent and To apply Green's theorem, the integral can be considered phosphorescent materials, dyes that bind to cell membranes, in the form of ÖO/öX-6P/öy and appropriate functions can be optical probes that preferentially accumulate in blood or in devised, e.g., P(x, y) and Q(x, y). For an area calculation, the intracellular space, phase resonance dye pairs, and the consider Q(x, y)=0 so that Ody=0, and let P=-y so that like. Detectors appropriate for use with Such contrast enhancing agents are Well known in the art. 8P/öy=-1. Then, the area can be calculated as follows: Numerous devices for acquiring, processing and display 35 ing data representative of one or more geometrical and/or optical properties of a cell Sample population in culture or an area of interest in Situ in an animal model may be employed. = foo/ox-Ploy didy One preferred device is a camera that acquires images of one or more areas of interest at predetermined time intervals that 40 can be compared to identify areas of changes in geometrical and/or optical properties that indicate physiological activity (Green's Theorem) or dysfunction. The data acquisition device preferably incor porates or is used in conjunction with a device that magnifies = fray the area of interest, Such as a microScope. 45 Magnification sufficient to provide resolution of indi =-yds vidual cells is preferred. An inverted microscope Such as a Nikon Diophot 300 is suitable. For high throughput screen ing techniques using cell Sample populations maintained under culture conditions, Samples in optically transparent 50 containerS Such as flaskS, plates and multi-Well plates, may be placed on an automated Stage that is controlled and moved in a programmed fashion to permit periodic exami nation of individual cells or cell populations according to a programmed Schedule. For example, a multi-Well culture 55 plate having a plurality of cell Samples may be placed on an For Volume calculations, conventional edge detection automated and controllable microscope Stage. The Stage is using, for example, a Sobel operator, can be used to focus controlled by an automated microcontroller So that it auto through an individual cell, which can be divided into a matically moves into position over each culture well. A data plurality (n) of individual, planar Sections. The Volume for Set relating to geometrical and/or optical properties of indi each of the “n” Sections can be calculated as AV;=AZ, AA; 60 vidual cells or a cell population is acquired for each position. and the Volume of the entire cell can be approximated as: In this manner, the System can rapidly and Systematically V=XAV. The determination and comparison of cell areas acquire data corresponding to many Samples. The physi and Volumes is preferably accomplished using computer ological environment in Selected wells may be altered by hardware and/or Software implementations. exposure to a physiological challenge, test agent or test According to another embodiment, methods and Systems 65 condition, and the System may continue to automatically of the present invention acquire and compare data represen acquire data from the same wells in each culture plate at tative of one or more optical properties of individual cells or predetermined time intervals following treatment, with data US 6,319,682 B1 11 12 acquired from various treatment wells being compared to graphical format that highlights geometrical or optical dif data acquired from various control Wells or empirically ferences indicative of physiological changes. A preferred determined controls. technique for presenting and displaying comparison data is Acquisition of data representative of one or more geo in the form of Visual images or photographic frames corre metrical and/or optical properties preferably provides high sponding to Spatially resolved areas of interest. This format Spatial resolution as well, So that geometrical or optical data provides a visualizable spatial location (two- or three corresponding to a particular spatial location is acquired at dimensional) of a cell population being analyzed. To various time intervals for comparison. In this fashion, data enhance and provide better visualization of high contrast acquired from Single cells or highly localized areas of areas indicating changes in geometrical and/or optical prop interest in cell Sample populations is compared to provide erties indicative of physiological activity or dysfunction, reliable and highly-Sensitive information concerning the comparison data may be processed to provide an enhanced physiological State or condition of the Sample population. contrast grey Scale or even a color image. A look up table High Spatial resolution is provided, for example, by imple (“LUT") may be provided, for example, that converts the menting high resolution cameras and charge coupled devices grey Scale values for each pixel to a different (higher (CCDs). Apparatus Suitable for obtaining Such images have contrast) grey Scale value, or to a color value. Color values been described in the patents incorporated herein by refer 15 may map to a range of grey Scale values, or color may be ence and are more fully described below. The optical detec used to distinguish between positive-going and negative tor preferably provides images having a high degree of going geometrical or optical changes. In general, color Spatial resolution at a magnification Sufficient to detect converted imageS provide higher contrast images that high Single cells. Several images may be acquired at predeter light changes in optical properties representing mined time intervals and combined, Such as by averaging, to physiological activity, function or dysfunction. provide images for comparison. Systems of the present invention generally comprise an Various data processing techniques may be advanta illumination Source for illuminating the biological material, geously used to assess the data collected in accordance with an optical detector for acquiring data relating to a geometri the present invention. Comparison data may be assessed or cal or optical property of the biological material, and data presented in a variety of formats. Processing may include 25 Storage and analysis and output device(s) for Storing data averaging or otherwise combining a plurality of data Sets to relating to a geometrical or optical property of the biological produce control, Subsequent and various comparison data material, comparing various data sets, and/or control data Sets. Data may be converted from an analog to a digital form profiles, to generate comparison data relating to changes in for processing, and back to an analog form for display as an geometrical and/or optical properties indicative of changes image. Alternatively, data may be acquired, processed, in the physiological State of Sample populations and to analyzed, and output in a digital form. provide or display the output data in a useful format. Data processing may also include amplification of certain An emir Source is used for illuminating an area of interest Signals or portions of a data Set (e.g., areas of an image) to during acquisition of data representing one or more dimen enhance the contrast Seen in data Set comparisons, and to Sional or intrinsic optical properties of cells or tissue at an thereby identify cells or cell populations undergoing 35 area of interest. The emir Source may be utilized to illuminate changes in geometrical and/or optical properties with a high an area of interest directly, as when in Vitro cell cultures degree of Spatial resolution. For example, according to one maintained in optically transparent containers are illumi embodiment, images are processed using a transformation in nated or when tissue is exposed, Such as in connection with which image pixel brightness values are remapped to cover Surgery, or it may be utilized to illuminate an area of interest a broader dynamic range of values. A "low value may be 40 indirectly through adjacent or overlying tissue Such as bone, Selected and mapped to Zero, with all pixel brightness values dura, Skin, muscle and the like. The emir Source employed in at or below the low value set to zero, and a “high value may the present invention may be a high or low intensity Source, be selected and mapped to a Selected value, with all pixel and may provide continuous or non-continuous illumination. brightness values at or above the high value mapped to the Suitable illumination Sources include high and intensity high value. Pixels having an intermediate brightness value, 45 Sources, broad Spectrum and non-chromatic Sources, representing the dynamic changes in brightness indicative of tungsten-halogen lamps, lasers, light emitting diodes, laser neuronal activity, may be mapped to linearly or logarithmi diodes, and the like. Polarized light sources and dark field cally increasing brightness values. This type of processing illumination techniques may also be used. Cutoff filters for manipulation is frequently referred to as a “histogram Selectively passing all wavelengths above or below a Stretch' and can be used according to the present invention 50 Selected wavelength may be employed. A preferred cutoff to enhance the contrast of data Sets, Such as images, repre filter excludes all wavelengths below about 695 nm. Senting changes in neuronal activity. Preferred emir wavelengths for acquiring data relating to Data processing techniques may also be used to manipu intrinsic optical Signals include, for example, wavelengths of late data Sets to provide more accurate combined and from about 450 nm to about 2500 nm, and most preferably, comparison data. For example, for in Vivo applications, 55 wavelengths of the near infrared spectrum of from about 700 movement, respiration, heartbeat, Seizure or refleX activity nm to about 2500 nm. Generally, longer wavelengths (e.g., may shift an area of interest during data acquisition. It is approximately 800 nm) are employed to detect cellular or important that corresponding data points in data Sets are tissue condition of locations beneath the Surface of cells or Spatially resolved and precisely aligned to provide accurate tissue, or beneath other materials Such as Skin, bone, dura, combined and comparison data. Optical markers may be 60 and the like cortical activity. Selected wavelengths of emr fixed at an area of interest and detected as the data is may also be used, for example, when various types of collected to aid in manual alignment or mathematical contrast enhancing agents are administered. The emir Source manipulation of data Sets. Various processing techniques are may be directed to the area of interest by any appropriate described below and in the patents incorporated herein by means. For Some applications, the use of optical fiberS is reference. 65 preferred. One preferred arrangement provides an emir Comparison data may be displayed in a variety of ways. Source through Strands of fiber optic using a beam splitter Comparison data may be displayed, for example, in a controlled by a D.C. regulated power Supply (Lambda, Inc.). US 6,319,682 B1 13 14 The optical detection methods of the present invention arranged in any Suitable arrangement. Specialized detectors may also usefully employ non-continuous illumination and for detecting Selected optical properties may be employed. detection techniques. For example, short pulse (time One preferred optical detector for acquiring data in the domain), pulsed time, and amplitude modulated (frequency format of an analog video Signal is a CCD Video camera domain) illumination Sources may be used in conjunction 5 which produces an output video signal at 30 HZ having, for with suitable detectors (see, Yodh, A. and Chance, B., example, 512 horizontal lines per frame using Standard RS Physics Today, March, 1995). Frequency domain illumina 170 convention. One Suitable device is a CCD-72 Solid State tion Sources typically comprise an array of multiple Source Camera (Dage-MTI Inc., Michigan City, Ind.). Another elements, Such as laser diodes, with each element modulated Suitable device is a COHU 6510 CCD Monochrome Camera at 180 out of phase with respect to adjacent elements (see, with a COHU 6500 electronic control box (COHU Chance, B. et al., Proc. Natl. Acad. Sci. USA, 90:3423–3427, Electronics, San Diego, Calif.). In Some cameras, the analog 1993). Two-dimensional arrays, comprising four or more Signal is digitized 8-bits deep on an ADI board (analog-to elements in two orthogonal planes, can be employed to digital board). The CCD may be cooled, if necessary, to obtain two-dimensional localization information. Such tech reduce thermal noise. niques are described in U.S. Pat. Nos. 4,972,331 and 5,187, 15 Data processing is an important feature of the optical 672 which are incorporated by reference herein in their detection and analysis techniques and Systems of the present entireties. invention. In use, for example, a CCD apparatus is prefer Time-of-flight and absorbance techniques (Benaron, D. A. ably adjusted (at the level of the analog signal and before and Stevenson, D. K., Science 259:1463–1466, 1993) may digitizing) to amplify the signal and spread the Signal across also be usefully employed in the present invention. In yet the full possible dynamic range, thereby maximizing the another embodiment of the present invention, a Scanning Sensitivity of the apparatus. Specific methods for detecting laser beam may be used in conjunction with a Suitable optical Signals with Sensitivity acroSS a full dynamic range detector, Such as a photomultiplier tube, to obtain high are described in detail in the patents incorporated herein by resolution data images, preferably in the form of an area of reference. Means for performing a histogram Stretch of the interest. 25 difference frames (e.g., Histogram/Feature Extractor HF Illumination with a part of the infrared spectrum allows 151-1-V module, Imaging Technology, Woburn, Mass.) may for detection of intrinsic optical Signals through tissue be provided, for example, to enhance each difference image overlying or adjacent the area of interest, Such as dura and acroSS its dynamic range. Exemplary linear histogram skull. One exemplary infrared emir Source Suitable for detec Stretches are described in Green, Digital Image Processing: tion of intrinsic optical Signals through tissue overlying or A Systems Approach, Van Nostrand Reinhold: New York, adjacent the area of interest is a Tunable IR Diode Laser 1983. A histogram Stretch takes the brightest pixel, or one from Laser Photonics, Orlando, Fla. When using this range with the highest value in the comparison image, and assigns of far infrared wavelengths, the optical detector is preferably it the maximum value. The lowest pixel value is assigned the provided as an infrared (IR) detector. IR detectors may be minimum value, and every other value in between is constructed from materials. Such as indium arsenide, germa 35 assigned a linear value (for a linear histogram Stretch) or a nium and mercury cadmium telluride, and are generally logarithmic value (for a log histogram Stretch) between the cryogenically cooled to enhance their Sensitivity to Small maximum and minimum values. This allows the comparison changes in infrared radiation. One example of an IR detec image to take advantage of the full dynamic range and tion System which may be usefully employed in the present provide a high contrast image that clearly identifies areas of invention is an IRC-64 infrared camera (Cincinnati 40 neuronal activity or inactivity. Electronics, Mason, Ohio). Noise (such as 60 Hz noise from A.C. power lines) is The area of interest is preferably evenly illuminated to filtered out in the control box by an analog filter. Additional effectively adjust the Signal over a full dynamic range, as adjustments may further enhance, amplify and condition the described below. Nonuniformity of illumination is generally analog signal from a CCD detector. One means for adjusting caused by fluctuations of the illumination Source and inten 45 the input analog signal is to digitize this signal at Video sity variations resulting from the three-dimensional nature of Speed (30 Hz), and view the area of interest as a digitized the tissue Surface. More uniform illumination can be pro image that is Subsequently converted back to analog format. Vided over the area of interest, for example, by using diffuse It is important that data, Such as consecutive data Sets lighting, mounting a wavelength cutoff filter in front of the corresponding to a particular area of interest, be aligned So optimal detector and/or emir Source, or combinations thereof. 50 that data corresponding to the same Spatially resolved loca Fluctuation of the illumination source itself is preferably tion is compared. If data Sets are misaligned prior to prevented by using a light feedback mechanism to regulate comparison, artifacts are introduced and the resulting com the power Supply of the illumination Source. In addition, a parison data Set may amplify noise and edge information. Sterile, optically transparent plate may contact and cover an Data misalignment may be caused by Sample movement or area of interest to provide a flatter, more even contour 55 motion, heartbeat, respiration, and the like. Large move Surface for detection. Fluctuations in illumination can be ments of cells in an area of interest being analyzed may compensated for using detection processing algorithms, require a new orientation of the detector. It is possible to including placing a constant shade grey image marker point compensate for Small movements of cells in the area of at the area of interest as a control point. interest by either mechanical or computational means, or a The System also comprises an optical detector for acquir 60 combination of both. ing a signal representative of one or more optical properties Real-time motion compensation and geometric transfor of the area of interest. Any photon detector may be mations may also be used to align corresponding data. employed as an optical detector. Suitable optical detectors Simple mechanical translation of data or more complex (and include, for example, photo diodes, photo multiplier tubes, generally more accurate) geometric transformation tech photo Sensitive Silicon detector chips, Such as those provided 65 niques can be implemented, depending upon the input data in CCD devices, and the like. Multiple emir sources and/or collection rate and amount and type of data processing. For multiple photon detectors may be provided and may be many types of image data, it is possible to compensate by a US 6,319,682 B1 15 16 geometrical compensation which transforms the images by of optical detectors via a variable Scan interface. A daughter translation in the X-y plane. In order for an algorithm Such board may Support the interfacing needs of many different as this to be feasible, it must be computationally efficient types of optical detectors and Supply variable Scan Signals to (preferably implementable in integer arithmetic), memory the acquisition motherboard. Preferably, the unit comprises efficient, and robust with respect to changes in ambient light. a daughter board interfacing to an RS-170A Video signal to For example, functional control points or numbers can be Support a wide base of cameras. Other camera types, Such as located in an area of interest and triangulation-type algo Slow Scan cameras with a higher spatial/contrast resolution rithms used to compensate for movements of these control and/or better Signal to noise ratio, can be developed and points. Goshtasby (“Piecewise Linear Mapping Functions incorporated in the inventive device, as well as improved for Image Registration,” Pattern Recognition 19:459-66, daughter boards to accommodate Such improved cameras. 1986) describes a method whereby an image is divided into Data relating to dimensional and/or intrinsic optical prop triangular regions using control points. A separate geometri erties of a Sample population acquired, for example, as cal transformation is applied to each triangular region to analog video signals, may be continuously processed using, Spatially register each control point to a corresponding for example, an image analyzer (e.g., Series 151 Image triangular region in a control image. 15 Processor, Imaging Technologies, Inc., Woburn, Mass.). An “Image warping” techniques may be employed whereby image analyzer receives and digitizes an analog video signal each Subsequent image is registered geometrically to the with an analog to digital interface and performs at a frame averaged control image to compensate for movement. Image speed of about 1/30th of a second (e.g., 30 Hz or “video warping techniques (described in, for example, Wolberg, Speed”). Processing the signal involves first digitizing the Digital Image Warping, IEEE Computer Society Press: Los Signal into a Series of pixels or Small Squares assigned a Alamitos, Calif., 1990), may be used. Image warping tech value (in a binary System) dependent upon the number of niques can further indicate when movement has become too photons (i.e., quantity of emr) being reflected off tissue from great for effective compensation and a new averaged control the part of the area of interest assigned to that pixel. For image must be acquired. example, in a Standard 512x512 image from a CCD camera, The data Storage processing and analysis function is 25 there would be 262,144 pixels per image. In an 8bit System, generally performed and controlled by a host computer. The each pixel is represented by 8 bits corresponding to one of host computer may comprise any general computer (Such as 256 levels of grey. an IBM PC type with an Intel 386, 486, Pentium or similar The Signal processor may include a programmable look microprocessor or Sun SPARC) that is interfaced with the up table (e.g., CM150-LUT16, Imaging Technology, emir Source and/or optical detector and controls data acqui Woburn, Mass.) initialized with values for converting grey Sition and flow, comparison computations, analysis, output, coded pixel values, representative of a black and white and the like. The host computer thus controls acquisition and image, to color coded values based upon the intensity of analysis of data and provides a user interface. each grey coded Value. Using image Stretching techniques, The host computer may comprise a single-board embed the highest and lowest pixel intensity values representing ded computer with a VME64 interface, or a standard (IEEE 35 each of the pixels in a digital image frame are determined 1014-1987) VME interface, depending upon bus band width over a region of the image frame which is to be stretched. considerations. Host computer boards which may be Stretching a Selected region over a larger range of values employed in the present invention include, for example, permits, for example, easier identification and removal of Force SPARC/CPU-2E and HP9000 Model 7471. The user relatively high, Spurious values resulting from noise. interface can be, for example, a Unix/X-Window environ 40 The Signal processor means may further include a plural ment. The image processing board can be, for example, ity of frame bufferS having frame Storage areas for Storing based upon Texas Instruments MVP and other chips to frames of digitized image data received from the A/D provide real-time image averaging, registration and other interface. The frame Storage area comprises at least one processing necessary to produce high quality difference megabyte of memory Space, and preferably at least 8 mega images for intraoperative Viewing. This board will also drive 45 bytes of Storage Space. An additional 16-bit frame Storage a 120x1024 RGB display to show a sequence of difference area may be provided as an accumulator for Storing pro images over time with pseudo-color mapping to highlight cessed image frames having pixel intensities represented by tumor tissue. Preferably, a second monitor is used for the more than 8bits. The processor means preferably includes at host computer to increase the overall Screen real estate and least three frame buffers, one for Storing the averaged Smooth the user interface. The processing board (fully 50 control image, another for Storing the Subsequent image, and programmable) can Support a VME64 master interface to a third for Storing a comparison image. control data transactions with the other boards. Lastly, a The Signal processor may further comprise an arithmetic peripheral control board can provide electrical interfaces to logic unit (e.g., ALU-150 Pipeline Processor) for performing control mechanical interfaces from the host computer. Such arithmetical and logical functions on data located in one or mechanical interfaces can include, for example, the light 55 more frame buffers. An ALU may, for example, provide Source and optical detector control box. image (data) averaging in real time. A newly acquired A real-time data acquisition and display System, for digitized image may be sent directly to the ALU and example, may comprise four boards for acquisition, image combined with control images stored in a frame buffer. A 16 processing, peripheral control and host computer. A minimal bit result can be processed through an ALU, which will configuration with reduced processing capabilities may 60 divide this result by a constant (i.e., the total number of comprise just the acquisition and host computer boards. The images). The output from the ALU may be stored in a frame acquisition board comprises circuitry to perform real-time buffer, further processed, or used as an input and combined averaging of incoming Video frames and allow readout of with another image. averaged frames at a maximum rate bus. A VME bus is Normally, areas of increased physiological activity preferred because of its high peak bandwidth and compat 65 exhibit an increase of the emir absorption capacity of the cell ibility with a multitude of existing VME products. The Sample or tissue (i.e., the cell Sample gets darker if visible acquisition board should also Support many different types light is used for emir illumination, or an intrinsic Signal US 6,319,682 B1 17 18 increases in a positive direction). Similarly, a reduction in pentium PC running windows NT. Image analysis algo physiological activity generally corresponds to a decrease of rithms may be written in C using Microsoft VisualC++ emir absorption capacity of the tissue (i.e., the tissue appears Version 5.0 compiler. For more rapid online processing, the brighter, or intrinsic Signals become negative). For example, data may be routed to dedicated imaging hardware residing data Set A is a Subsequent averaged image and data Set B is in the PC computer. For example, IM-PCI hardware (by an averaged control image. Normally, when a pixel in data Imging Technology Inc., Bedford, Mass.) could be used. Set A is Subtracted from a pixel in data Set B and a negative One Such configuration would consist of the following value results, this value is treated as Zero. Hence, difference IM-PCI boards and modules: IM-PCI, AMVS, and a images cannot account for areas of inhibition. The present CMALU invention provides a method for identifying both negative The imaging methods applied to in Vivo applications may and positive intrinsic signals, by: (a) Subtracting data Set A acquire data at the Surface of an area of interest. AS described (a Subsequent averaged image) from data set B (an averaged above, longer wavelengths of emr (in the infrared range) can control image) to create a first difference data Set, whereby be used to image areas of interest which are deeper in tissue all negative pixel values are Zero; and (b) Subtracting data set or below overlying tissue. In Some areas of the body longer B from data Set A to create a Second difference data Set 15 wavelength visible light and near infrared emir can easily whereby all negative pixel values are Zero; and adding the pass through Such tissue for imaging. Moreover, if a differ first and Second difference data Sets to create a “Sum ence image is created between the image acquired at 500 nm difference data set.” The Sum difference data set shows areas emir and the image acquired at 700 nm emir, the difference of increased activity (i.e., color coded with warmer colors image will show an optical Slice of tissue. Administration of Such as yellow, orange, red) and may be visualized as image an imaging agent which absorbs Specific wavelengths of emr areas of less activity or inhibition (i.e., color coded with can act as a tissue filter of emr to provide a filter in the area colder colors Such as green, blue, purple). Alternatively, one of interest. In this instance, it is desirable to utilize an can overlay the first difference data Set on the Second imaging agent that remains in the tissue for a prolonged difference data set. The difference output may be visualized period of time. as an image and may be Superimposed on the real time 25 In a simple System Suitable for assessing cell populations analog image to provide an image of the area of interest in Vivo in animal or tissue culture models, the Systems of the (e.g., cortical Surface) Superimposed with a color-coded present invention may include one or more optical fiber(s) difference frame to indicate where there are intrinsic signals operably connected to an emir Source that illuminates cells or in response to a challenge, Stimulus, paradigm, or the like. tissue, and another optical fiber operably connected to an The comparison (e.g., difference) data may be further optical detector, Such as a photodiode, that detects one or processed to Smooth out the data and remove high frequency more optical properties of the illuminated cells or tissue. The noise. For example, a lowpass spatial filter can block high detector may be used to acquire obtain control data repre Spatial frequencies and/or low Spatial frequencies to remove Senting the “normal” or “background” optical properties of high frequency noises at either end of the dynamic range. a Sample population, and then to acquire Subsequent data This provides a Smoothed-out processed difference data Set 35 representing the optical properties of the Sample population (in digital format). The digitally processed difference data during or following administration of a test agent or test Set can be provided as an image and color-coded by assign condition. A physiological challenge and/or a stimulus that ing a spectrum of colors to differing shades of grey. This Stimulates a disease or pathological State may be adminis image may then be converted back to an analog image (by tered prior to administration of the treatment agent or an ADI board) and displayed for a real time visualization of 40 condition. The System comprises or is in communication differences between an averaged control image and Subse with a data Storage and processing System having informa quent images. Moreover, the processed difference image can tion Storage and processing capability Sufficient to compare be Superimposed over the analog image to display Specific the geometrical and/or optical properties of individual cells tissue Sites where a contrast enhancing agent may have a or cell Samples to empirically determined Standards, or to faster uptake, or where an intrinsic Signal may be occurring. 45 data acquired at different points in time. Processing Speed may be enhanced by adding a real time In operation, an area of interest in an in vitro or in vivo modular processor or faster CPU chip to the image proces cell Sample is illuminated with electromagnetic radiation Sor. One example of a real time modular processor which (emir) and one or a Series of data points or data sets may be employed in the present invention is a 150 RTMP representing one or more geometrical and/or optical prop 150 Real Time Modular Processor (Imaging Technology, 50 erties of a Spatially resolved area of interest is acquired Woburn, Mass.). The processor may further include an during an interval of “normal” physiological activity. This optical disk for Storing digital data, a printer for providing a data represents a control, or background data profile for that hard copy of the digital and/or analog data and a display, particular cell Sample under those particular physiological Such as a Video monitor, to permit the user to continuously conditions. A Series of data Sets is preferably combined, for monitor the comparison data output. 55 example by averaging, to obtain a control data profile. The A Single chassis may house all of the modules necessary control data profile is Stored for comparison with other data to provide optical detection and analysis in a format that can Sets. Similarly, control data Sets may be collected and Stored be easily interpreted, Such as an image format, according to that represent a background data profile for particular cell the present invention. The necessary components, whether types under Specified physiological conditions. or to whatever degree integrated, may be installed on a rack 60 Data Sets representing the corresponding geometrical and/ that is easily transportable, along with display monitors and or optical property of the Sample population at the Same, peripheral input and output devices. Spatially resolved areas of interest, are acquired during a A preferred high resolution and high performance System Subsequent time period. For monitoring applications, data comprising a PentaMAX 576x384FT LCD system (by Prin may be collected at regular time intervals to monitor the ceton Instruments Inc., N.J.) digitizes the data at the chip and 65 condition of the cell Sample and to detect aberrations from provides a large dynamic range and reduced noise. This the baseline profile. For Screening applications, one or more system may be interfaced using a PCI-bus to a dual-400 Mhz. Subsequent data set(s) is collected during a period following US 6,319,682 B1 19 20 physiological activity or inhibition, induced, for example, by enhanced image. A Series of images, two Seconds apart, were introduction of a test compound or by exposure to a test acquired and the average value was calculated for each condition. Physiological activity or inhibition may be image and plotted as a point on the graph. The tissue was induced by a “natural occurrence Such as a Seizure or Stroke electrically Stimulated for two Seconds at the points indi in an animal model, or it may be induced by administering cated by the Straight lines. The Small peak indicates the a paradigm or an agent to an in vitro or in Vivo cell Sample maximum optical change induced by the first Small electrical to Stimulate changes in geometrical and/or optical properties Stimulation, the larger peak from the Second larger Stimulus. of the cell Sample that are indicative of physiological The electrical Stimulation was ceased after two Seconds and activity or inhibition. During a monitoring interval or Stimu the tissue was allowed to recover. The plots of the recovery lation of an intrinsic physiological response, one or a Series are characteristic of the ion-homeostatic mechanisms of the of Subsequent data Sets, representing one or more of the tissue. Their recovery could be quantified, for example, by detected geometrical or optical properties of the area of finding the best exponential fits for the recovery periods. interest, is acquired. A Series of Subsequent data Sets is Methods and systems of the present invention are well preferably combined, for example by averaging, to obtain a Suited for high throughput Screening of libraries of Subsequent data Set. The Subsequent data Set is compared 15 compounds, and combinations of compounds, to identify with the control data Set to obtain a comparison data Set, candidate compounds and combinations having desired preferably a difference data Set. Comparison data Sets are therapeutic or diagnostic properties. In one approach, well then analyzed for evidence of changes in geometrical and/or established agents, Such as therapeutic agents effective in optical properties representative of physiological activity or treating various conditions are identified; modifications to inhibition within the area of interest. the therapeutic agent(s) are made, for example, analog and FIG. 1 shows a Schematic flow diagram illustrating an derivative compounds are identified and Synthesized; and exemplary System, as well as exemplary output data of the the modified agents are then Screened for efficacy under present invention with reference to in vitro cell populations. various conditions using Screening techniques of the present A cell population may comprise cells in Suspension at a invention. In a similar fashion, combinations of therapeutic sparse cell density, or confluent layers of cells, or layers of 25 compounds, combinations of a therapeutic compound with cells at other predetermined cell densities, or a tissue candidate agents, and combinations of candidate agents may Sample, Such as a tissue Slice. Maintenance of a wide variety be Screened for efficacy under certain conditions using of cell and tissue Samples under cell culture conditions is Screening techniques of the present invention well known in the art. Numerous agents are known for treatment of various The Sample population is placed at a predetermined conditions, Such as cancers and other proliferative disorders, location on a platform Such as on a microScope Stage. In the psychiatric and neurological disorders, chronic pain, and example shown in FIG. 1, the Sample is an acute rat various other conditions. In one approach, Such agents may hippocampal Slice maintained in a Submerged perfusion be Screened using techniqueS of the present invention. chamber. Alternatively, the Sample may be cell Samples Modifications to Such agents may then be made, for maintained in cell culture media in flasks, multiple well 35 example, using combinatorial chemistry and molecular plates, and the like. Multiple well tissue culture plates may modeling techniques, to produce modified candidate agents, be used for high throughput Screening, in combination with which may be Screened for various activities using methods an automated Stage for positioning cell Samples in individual of the present invention. Similarly, combinations of both Wells for optical detection at predetermined intervals. known agents and modified agents may be Screened using Programmable, automated positioning devices are well 40 techniques of the present invention. known in the art. The present invention contemplates high throughput An optical detector (in this case, a CCD camera) is Screening of agents for treating cancers and other prolifera attached to the camera-port of the microscope. During one tive disorders. The following agents, and modifications of or more control period(s) and one or more test period(s), the following agents, including analogs, derivatives, data relating to dimensional and/or optical properties of 45 fragments, active moieties, and the like, may be Screened individual cells or of an area of interest in the cell Sample are using methods and Systems of the present invention: AA acquired, Stored and processed. Acquisition and processing (ara-C, Adriamycin), AAF (2-acetylaminofluorene), AAFC of data may be accomplished as described below and in the (fluro citabine); ABC (Adriamycin, BCNU, Examples. ); ABCD (Adriamycm, , The grey-Scale image on the upper right is the unproc 50 CCNU and ); ABCM (Adriamycin, bleomycin, essed image of the tissue-slice as viewed by the CCD cyclophosphamide, mitomycin-C); ABD (Adriamycin, camera. This slice was then electrically Stimulated at two bleomycin, DTIC); ABDIC (Adriamycin, bleomycin, different intensities: a low-intensity electrical Stimulus caus dacarbazine, CCNU and prednisone); ABDV (Adriamycm, ing a Small increase in neuronal and Synaptic activation; and bleomycin, DTIC, ); ABLC (amphotericin B lipid a high-intensity electrical Stimulus causing a larger increase 55 complex); ABOS (, bleomycin sulfate, in neuronal and Synaptic activity. , Streptozocin); ABP (Adriamycin, bleomycin, The Stim-Control image was generated as described prednisone); ABPP (bropirimine); ABV (Actinomycin-D, below in Example 1. Briefly, an image acquired during the bleomycin, Vincristine); ABV (Adriamycm, bleomycin, electrical Stimulation was Subtracted from an image acquired vinblastine); ABVD (Adriamycin, bleomycin, Vincristine, in the control state. This image was then filtered with a 60 dacarbazine); ABVE (Adriamycin, bleomycin, Vincristine, low-pass filter, histogram-Stretched, and contrast enhanced. ); ABVP (Adriamycin, bleomycin sulfate, Images may be pseudo-colored to indicate intensity of , prednisone); ABVP (Adriamycin, bleomycin activity-evoked optical change (arrow on bar). sulfate, vinblastine, prednisone); ABVP (Adriamycin, bleo The dynamic optical changes represented in these images mycin Sulfate, Vincristine, prednisone); AC (Adriamycin, can also be plotted as a graph (lower right image). Here, 65 ); AC (Adriamycin, CCNU); AC (Adriamycin, each data-point represents the average change in light ); AccuSite; Ac-D-Ac (Adriamycin, , transmission through the Small box indicated on the contrast Adriamycin); ACe (Adriamycin, cyclophosphamide); ACE US 6,319,682 B1 21 22 Adriamycin, cyclophosphamide, etoposide); ACFUCY ara-C+6TG (, thioguanine), ara-C-HU (ara-C, (actinomycin D, 5-, cyclophosphamide); ACID hydroxyurea); Ara-Cytidine (cytarabine), ara-G; Aredia (Adriamycin, cyclophosphamide, imidazole, ); (pamidronate disodium); Arensin (fadrozole); Arimidex Acivicin; ; aclarubicin HCl, ACM (Adriamycin, (anastrozole); Arotinoid; ASHAP (Adriamycin, Solu cyclophosphamide, ); ACNU (); Medrol high-dose ara-C, Platinol); ASHAP/BACOS; ASN ACOAP (Adriamycin, cyclophosphamide, Oncovin, (L-); ASP (L-asparaginase); asparaginase cytosine arabinoside, prednisone); ACOP (Adriamycin, (Elspar); ATG (antithymocyte globulin); Atzpodien regimen cyclophosphamide, Oncovin, prednisone); ACOPP for renal cell carcinoma, autolymphocyte therapy; AV (Adriamycin, cyclophosphamide, Oncovin, prednisone, (Adriamycin, Vincristine); AVAD (doxorubicin, Vincristine, ); ACR (aclarubicn); ACT (actinomycin); cytarabine, dexamethasone), Avicidin; AVM (Adriamycin, ACT-C (actinomycin-C); ACT-D (actinomycin-D); Actinex vinblastine, methotrexate); AVM (Adriamycin, Vincristine, (); actinomicin-D (ACT-D); ACT-FU-Cy mitomycin-C); AVP (actinomycin D, Vincristine, Platinol); (actinomycin-D, 5-FU, cyclophosphamide); ADBC AVP (Adriamycin, Vincristine, procarbazine); ; (Adriamycin, DTIC, bleomycin, CCNU); ADE (ara-C, 5-azacitidine (Mylosar); azathioprine (Imuran); AZC daunorubicin, etoposide); adenine arabinoside (Ara-A); 15 (azacitidine); aziridinylbenzoquinone (diaziquone); AZQ ADIC (Adriamycin, DTIC), ADOC (Adriamycin, cisplatin, (aziridinylbenzoquinone); BAC (BCNU, cytarabine, Vincristine, cyclophosphamide); AdOAP (Adriamycin, cyclophosphamide); Bacatin III; BACO (bleomycin, Oncovin, ara-C, prednisone); AdOP (Adriamycin, Oncovin, Adriamycin, CCNU, Oncovin); BACOD (bleomycin, prodnisone); adozelesin; ADR-529; Adria + BCNU Adriamycin, cyclophosphamide, Oncovin, dexamethasone); (Adriamycin-i-BCNU); Adriamycin PFS; Adriamycin RDF; BACON (bleomycin, Adriamycin, CCNU, Oncovin, nitro Adria-L-PAM (Adriamycin, L-phenyl-alanine mustard); gen mustard); BACOP (bleomycin, Adriamycin, Adrucil (fluorouracil); AF 1890 ((); AFM cyclophosphamide, Oncovin, prednisone); BACT (BCNU, (Adriamycm, 5-fluorouracil, methotrexate); AGT ara-C, cyclophosphamide, 6-thioguanine); BACT (a minoglute thimide); Agrelin (an agrellide); AID (bleomycin, Adriamycim, cyclophosphamide, tamoxifen); (Adriamycin, , dacarbazine, mesna), AIM 25 BAM/BLITZ (B4 blocked ricin); BAMON (bleomycin, (L-asparaginase, ifosfamide, methotrexate); AL-721; Adriamycin, methotrexate, Oncovin, ); aldesleukin (Proleukin); alfa-2 or 26 interferon; alfacalcidol BAP (bleomycin, Adriamycin, prednisone); BAPP (One-Alpha); Alferon LDO; interferon alfa-n3); Alferon-N (bleomycin, doxorubicin, cisplatin, prednisone); BAVIP (interferon alfa-n3); Alkaban-AQ (vinblastine); Alkeran (bleomycin, Adrinamycin, vinblastine, imidazole (); all trans-retinoic acid (); Allergan 211 carboxamide, prednisone); BBVP-M (BCNU, bleomycin, (idoxuridine); ALOMAD (Adriamycin, Leukeran, Oncovin, VePesid, prednisone, methotrexate); BCAP (BCNU, methotrexate, actinomycin D, dacarbazine); Alpha-Beta cyclophosphamide, Adriamycin, prednisone); BCAVe (alpha tocopherol and beta caroteine); Alpha Chymar (alpha (bleomycin, CCNU, Adriamycin, Velban); BCD chymotrypsin); alpha interferon (IFN-A); alpha1 (bleomycin, cyclophosphamide, dactinomycin); BCG antitrypsin; alpha-2 interferon(IFN-alpha-2), 5-alpha reduc 35 (bacillus Calmette-Guerin vaccine); B-CHOP (bleomycin tase inhibitors; alpha-TGI (teroxirone); AlphaNine; ALTO Cytoxan, hydroxydaunomycin, Oncovin, prednisone); (afterloading tandem and ovoids); ALT-RCC BCMF (bleomycin, cyclophosphamide, methotrexate, (autolymphocyte-based treatment for renal cell carcinoma); fluorouracil); BCNU (bis-chloronitrosourea) (carmustine); (Hexalen, Hexastat); AmBisome; amethopterin BCOP (BCNU, cyclophosphamide, Oncovin, prednisone); (methotre Xate); a milo ride (Mid a mor); 40 BCP (BCNU, cyclophosphamide, prednisone); BCVP 9-aminocamptothecin, aminoglutethimide (Cytadren, AGT, (BCNU, cyclophosphamide, Vincristine, prednisone); Elipten); ; amiothiadiazole, AML-2-23 mono BCVPP (BCNU, cyclophosphamide, vinblastine, clonal antibody; AMMEN-OE5 monoclonal antibody; procarbazine, prednisone); BCVPP-bleo (BCNU, amonafide (nafidimide); amphotericin B colloidal dispersion cyclophosphamide, vinblastine, procarbazine, bleomycin); (ABCD); amphotericin B lipid complex (ABLC); Ampligen 45 BCX-34; B-DOPA (bleomycin, dacarbazine, Oncovin, (polyribonucleotide); AMSA (); amsacrine prednisone, Adriamycin); BEAC (BCNU, etoposide, ara-C, (m-AMSA, AMSA, Amsidyl); Amsidyl (amsacrine); anak cyclophosphamide); BEAM (BCNU, etoposide, cytarabine, inra (Antril); (Agrelin), ANAN (anandron); anan melphalan); BEMP (bleomycin, Eldisine, mitomycin, dron (nilutamide); anastrozole (Arimidex); Andro-Cyp; Platinol); BEP (bleomycin, etoposide, Platinol); Betaseron Android-F (**Note: Discontinued in 1991); Andronad (* * 50 (interferon beta-1b); BHD (BCNU, hydroxyurea, Note: Discontinued in 1991); Andronate (testosterone dacarbazine); BHDV (BCNU, hydroxyurea, dacarbazine, cypionate); Andropository (testosterone enanthate); Andryl Vincristine); bicalutamide; BiCNU (carmustine); B-IFN (testosterone); annamycin LF (liposomal annamycin); (beta-interferon); Biodel Implant/BCNU; BioTropin; BIP anthramycin (also antramycin); anthrapyrazole; anti-B4 (bleomycin, ifosfamide, Platinol); bisacetamide blocked ricin; anti-EGFR (RG 838520); anti-MY9-blocked 55 (heramethylene); bisantrene hydrochloride (Bisantrene); ricin; anti-T12 allogeneic BMT, anti-TAP-72 immunotoxin; Bisantrene (bisantrene hydrocbloride); bischlorethylnitro Antril (anakinra); AOPA (ara-C, Oncovin, prednisone, Surea (BCNU); bispecific antibody 520C9x22; bizelesin; asparaginase); AOPE (Adriamycin, Oncovin, prednisone, Blenoxane (bleomycin); BLEO (bleomycin); BLEO-COMF etoposide); APC (AMSA, prednisone, ); APE (bleomycin, cyclophosphamide, Oncovin, methotrexate, (Adriamycin, Platinol, etoposide); APE (ara-C, Platinol, 60 fluorouracil); BLEO-MOPP (bleomycin, nitrogen mustard, etoposide); APO (Adriamycin, prednisone, Oncovin); Onco Vin, procarbazine, prednisone); bleomycin AR-623 (liposomal Tretinoin), ara-A (adenine arabinoside); (Blenoxane) (BLEO) (BLM); bleomycin HCl; bleomycin ara-AC (azacytosine arabinoside) (fazarabine), arabinofura sulfate; BLITZ (monoclonal antibodies); BLM (bleomycin nosylcytosine (cytarabine); arabinosyl cytosine sulfate); BM-92 103; B-MOPP (ble o mycin, (cytarabine), ara-C (cytosine arabinoside) (cytarabine), ara 65 mechlorethamine, Oncovin, procarbazine, prednisone); C+ADR (cytarabine, Adriamycin); ara-C+DNR--PRED--MP BMP (BCNU, methotrexate, procarbazine); BMS-182248 (cytarabine, daunorubicin, prednisolone, ); (BR96-Dox); BMY-28090; BMY-45622; BOAP US 6,319,682 B1 23 24 (bleomycin, Oncovin, Adriamycin, prednisone); BOLD and amp; injections; CAV (cyclophosphamide, Adriamycin, (bleomycin, Oncovin, , dacarbazine); BOMP Velban); CAV (cyclophosphamide, Adriamycin, (bleomycin, Oncovin, Matulane, prednisone); BONP Vincristine); CAVe, CA-Ve (CCNU, Adriamycin, Velban); (bleomycin, Oncovin, Natulan, prednisolone); BOP (BCNU, CAVP (cyclophosphamide, Adriamycin, VM-26, Oncovin, prednisone); BOP (bleomycin, Oncovin, Platinol); prednisone); CAVP-I (cyclophosphamide, Adriamycin, BOPAM (bleomycin, Oncovin, prednisone, Adriamycin, Vincristine, prednisone); CAVP-16 (cyclophosphamide, mechlorethamine, methotrexate); BOPP (BCNU, Oncovin, Adriamycin, VP-16); CAVPM (cyclophosphamide, procarbazine, prednisone); Borocell; BR-96 (doxorubicin Adriamycin, VP-16, prednisone, methotrexate); CB10-277; monoclonal antibody immunoconjugate); brachytherapy; CPBBA (cyclophosphamide, bleomycin, procarbazine, BRCA genetic testing, brequinar, brequinar Sodium; prednisone, Adriamycin); CBV (cyclophosphamide, BCNU, BRL39 123 A; bro mode oxyuridine (BU dR); VePesid); CBV (cyclophosphamide, BCNU, VP-16); CBVD 5-bromodeoxyuridine (broXuridine or BUdR); 5-bromo-2- (CCNU, bleomycin, vinblastine, dexamethasone); CC deoxyuridine, 5-bromouracil; bromfenac, bropirimine; (, cyclophosphamide); CC49 monoclonal anti broxuridine (BUdR); BSO (buthionine sulfoximine); BSRL body; CCAVV (CCNU, cyclophosphamide, Adriamycin, (buserelin) (Suprefact); BT (BCNU, triazinate); BUdR 15 Vincristine, VP-16); CCFE (cyclophosphamide, cisplatin, (bromodeoxyuridine or broxuridine); BU (); BUS 5-fluorouracil, estramustine); CCM (cyclophosphamide, (busulfan, Myleran); buserelin (BSRL) (Suprefact); busul CCNU, methotrexate); CCMA (CCNU, cyclophosphamide, fan (BU, Myleran); buthionine sulfoximine (BSO); BVAP methotrexate, Adriamycin); CCNU (lomustine); CCNU-OP (BCNU, Vincristine, Adriamycin, prednisone); BV-ara-U; (CCNU, Oncovin, prednisone); CCOB (CCNU, BVCPP (BCNU, vinblastine, cyclophosphamide, cyclophosphamide, Oncovin, bleomycin); CCV (CCNU, procarbazine, prednisone); BVD (BCNU, Vincristine, cyclophosphamide, Vincristine); CCV-AV (CCNU, dacarbazine); BVDS (bleomycin, Velban, doxorubicin, cyclophosphamide, Vincristine/Adriamycin, Vincristine); streptozocin); BVPP (BCNU, Vincristine, procarbazine, CCVB (CCNU, cyclophosphamide, Vincristine, prednisone); BW301U (piritrexim); BW A770U (crisnatol bleomycim); CCVPP (CCNU, cyclophosphamide, Velban, mesylate); CA 15-3; CA 72-4; CA (cyclophosphamide, 25 procarbazine, prednisone); CCVV (cyclophosphamide, Adriamycin); CABOP, CA-BOP (cyclophosphamide, CCNU, VP-16, vincristine); CCVVP (cyclophosphamide, Adriamycin, bleomycin, Oncovin, prednisone); CABS CCNU, VP-16, vincristine, Platinol); CD (cytarabine, (CCNU, Adriamycin, bleomycin, streptozocin); CAC daunorubicin); CdA (chlorodeoxyadenosine) (); (cisplatin, ara-C, caffeine); CACP (cisplatin); cactinomycin 2-CdA (2-chlorodeoxyadenosine); CDC (carboplatin, (actinomycin C); CAD (cyclophosphamide, Adriamycin, doxorubicin, cyclophosphamide); CDDP, C-DDP (cis dacarbazine); CAD (cytosine arabinoside, daunorubicin); diamminedichloroplatinum); CDDP (cisplatin); CDE CADIC (cyclophosphamide, Adriamycin, DTIC), CAE (cyclophosphamide, doxorubicin, etopoSide); CEAker; CEB (cyclophosphamide, Adriamycin, etoposide); CAF (carboplatin, etoposide, bleomycin); CECA (cisplatin, (cyclophosphamide, Adriamycin, 5-fluorouracil); CAFFI etoposide, cyclophosphamide, Adriamycin); CeeNu (CCNU (cyclophosphamide, Adriamycin, 5-fluorouracil by continu 35 or lomustine), CEF (cyclophosphamide, , ous infusion); CAFP (cyclophosphamide, Adriamycin, 5-fluorouracil); cellogovab (OncoScint OV103); CEM 5-fluorouracil, prednisone); CAFTH (cyclophosphamide, (cytosine arabinoside, etoposide, methotrexate); Centixsin; Adriamycin, 5-fluorouracil, tamoxifen, Halotestin); CAFVP centrifugation; CEP (CCNU, etoposide, ); (cyclophosphamide, Adriamycin, 5-fluorouracil, Vincristine, CEP (cyclophosphamide, etoposide, Platinol); Ceptrate SC prednisone); CALF (cyclophosphamide, Adriamycin, leuco 40 (monoclonal antibodies); Cerubidine (daunorubicin); CEV V or in calcium , 5-flu or our acil); CALF-E; (cyclophosphamide, etoposide, Vincristine); CF (cisplatin, (cyclophosphamide, Adriamycin, leucovorin calcium, 5-fluorouracil); CF (citrovorum factor); CF (leucovorin); 5-fluorouracil, ethinyl estradiol); calusterone; CAM CFL (cisplatin, 5-fluorouracil, leucovorin calcium); CFM (cyclophosphamide, Adriamycin, methotrexate); CAMB (cyclophosphamide, 5-fluorouracil, ); CFP (cyclophosphamide, Adriamycin, methotrexate, bleomycin); 45 (cyclophosphamide, 5-fluorouracil, prednisone); CFPT CAMELEON (cytosine arabinoside, high-dose (cyclophosphamide, 5-fluorouracil, prednisone, tamoxifen); methotrexate, leucov orin, Oncovin); CAMEO CGS 16949 A; CHAD (cyclophosphamide, (cyclophosphamide, Adriamycin, methotrexate, etoposide, hexamethylmelamine, Adriamycin, DDP); CHAMOCA Oncovin); CAMF (cyclophosphamide, Adriamycin, (Cytoxan, hydroxyurea, actinomycin D, methotrexate, methotrexate, 5-fluorouracil); CAME (cyclophosphamide, 50 Oncovin, calcium; folinate, Adriamycin); CH1 VPP, Adriamycin methotrexate, folic acid); CAMLO (cytosine Ch1 VPP (chlorambucil, vinblastine, procarbazine, arabinoside, methotrexate, leucovorin, Oncovin); CAMP prednisone); CHAP (cyclophosphamide, Hexalen, (cyclophosphamide, Adriamycin, methotrexate, Adriamycin, Platinol); CHD (cyclophosphamide, procarbazine); Campath 1H; -11 (CPT-11); hexamethylmelamine, DDP); CHD-R (cyclophosphamide, CAO (cyclophosphamide, Adriamycin, Oncovin); CAP 55 hexamethylmelamine, DDP, radiotherapy); CHEX-UP (cyclophosphamide, Adriamycin, Platinol); CAP (cyclophosphamide, hexamethylmelamine, 5-fluorouracil, (cyclophosphamide, Adriamycin, prednisone); CAP-I Platinol); CHF (cyclophosphamide, hexamethylmelamine, (cyclophosphamide, Adriamycin, Platinol); CAP-II 5-fluorouracil); chimeric 17-1A (C17-1A); chimeric L6 (cyclophosphamide, Adriamycin, high-dose Platinol); CAP monoclonal antibodies; CHIP (cis-dichlorotranshydroxy BOP (cyclophosphamide, Adriamycin, procarbazine, 60 bis-isopropylamine platinum IV); CHIP (iproplatin); bleomycin, Oncovin, prednisone); capiromab pendetide; chlorambucil (CLB or Leukeran); Chlorbutin CAPPr (cyclophosphamide, Adriamycin, Platinol, (chlorambucil); ; chlormethine HCl; prednisone); carace mide; carbetimer; carboplatin 8-chlorocamp; 2-chlorodeoxyadenosine (2-CdA), chlorode (Paraplatin); carboxamide; Cardioxane; carmustine (BCNU) oxyadenosine; chlorotrianisene (Tace); chlorozotocin (BiCNU); carubicin; carubicin HCl; carzelesin; Casodex; 65 (DCNU); CHL+PRED (chlorambucil, prednisone); Chl CAT (cytosine arabinoside, Adriamycin, 6-thioguanine); VPP (chlorambucil vinblastine, procarbazine, prednisone); CAT (cytosine arabinoside, thioguanine); Catrix capsules CHO (cyclophosphamide, hydroxydaunomycin, Oncovin); US 6,319,682 B1 25 26 CHOB (cyclophosphamide, hydroxydaunomycin, Oncovin, ara-C); COMB (cyclophosphamide, Oncovin, methotrexate, bleomycin); CHOD (cyclophosphamide, bleomycin); COMB (cyclophosphamide, Oncovin, hydroxydaunomycin, Oncovin, dexamethasone); CHOP MeCCNU, bleomycin); COMBAP (cyclophosphamide, (cyclophosphamide, Halotestin, Oncovin, prednisone); Oncovin, methotrexate, bleomycin, Adriamycin,; CHOP (cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone); COMe (cyclophosphamide, Oncovin, prednisone); CHOP-BLEO (cyclophosphamide, methotrexate); COMET-A (cyclophosphamide, Oncovin, hydroxydaunomycin, Oncovin, prednisone, bleomycin); methotrexate, leucovorin, etoposide, ara-c); COMF CHOPE (cyclophosphamide, Halotestin, Oncovin, (cyclophosphamide, Oncovin, methotrexate, 5-fluorouracil); prednisone, etoposide); CHOR (cyclophosphamide, COMLA (cyclophosphamide, Oncovin, methotrexate, hydroxydaunomycin, Oncovin, radiotherapy); chromic leucovorin, ara-C); COMP (Cyclophosphamide, Oncovin, phosphate P 32; CHVP (cyclophosphamide, methotrexate, prednisone); conjugated estrogens, hydroxydaunomycin, VM-26, prednisone); CI-958, 973, CONPADRI, COPADRI-I (cyclophosphamide, Oncovin, 980, CIA (CCNU, ifosfamide, Adriamycin); cinchona bark; L-phenylalanine mustard, Adriamycin); Consensus inter CIS 39300; cis-retinoic acid (CRA); 13-cis-retinoic acid feron; Cooper CMFVP (cyclophosphamide, methotrexate, (13-CRA); CISCA, CisCA (cisplatin, cyclophosphamide, 15 5-fluorouracil, Vincristine, prednisone); Cooper Regimen Adriamycin); CISCAii/BViv (cisplatin, cyclophosphamide, (cyclophosphamide, methotrexate, 5-fluorouracil, Adriamycin, vinblastine, bleomycin); cis-DDP (cisplatin); Vincristine, prednisone); COP (cyclophosphamide, Oncovin, cis-diamminedichloroplatinum (cisplatin); cisplatin (CDDP predisone); COP (cyclophosphamide, Oncovin, or cis-platinum or DDP or Platinol); cisplatin/collagen predisolone); COPA (cyclophosphamide, Oncovin, matrix; citrovorum factor (CF); citrovorum rescue; CIVPP prednis one, Adria mycin); CO PA - BLEO (chlorambucil, vinblastine, procarbazine, prednisone); (cyclophosphamide, Oncovin, prednisone, Adriamycin, cladribine (Leustatin); ; CLB (chlorambucil); bleomycin); COPAC (CCNU, Oncovin, prednisone, CMC (cyclophosphamide, methotrexate, CCNU); CMC Adriamycin, cyclophosphamide); COP-B, COPB VAP (cyclophosphamide, methotrexate, CCNU, Vincristine, (cyclophosphamide, Oncovin, prednisone, bleomycin); Adriamycin, procarbazine); CMF (cyclophosphamide, 25 COP-BLAM (cyclophosphamide, Oncovin, prednisone, methotrexate, 5-fluorouracil); CMF-AV (cyclophosphamide bleomycin, Adriamycin, Matulane); COP-BLEO methotrexate, 5-fluorouracil, Adriamycin, Vincristine); (cyclophosphamide, Oncovin, prednisone, bleomycin); CMFAVP (cyclophosphamide, methotrexate, 5-fluorouracil, COPE (cyclophosphamide, Oncovin, Platinol, etoposide); Adriamycin, Vincristine, prednisone); CMF-BLEO COPP (CCNU, Oncovin, procarbazine, prednisone); COPP (cyclophosphamide, methotrexate, 5-fluorouracil, (cyclophosphamide, Oncovin, procarbazine, prednisone); bleomycin); CMF-FLU (cyclophosphamide, methotrexate, Cosmegen (dactinomycin); CP (Cytoxan, Platinol); CP 5-flu or our a cil, flu oxy me ster one); CMF H (cyclophosphamide, prednisone); CPA (cyclophosphamide); (cyclophosphamide, methotrexate, 5-fluorouracil, CPB (cyclophosphamide, Platinol, BCNU); CPC hydroxyurea); CMFP (cyclophosphamide, methotrexate, (cyclophosphamide, Platinol, carboplatin); CPDD (cis 5-fluorouracil, prednisone); CMFPT (cyclophosphamide, 35 platinum diamminedichloride); CPM (CCNU, procarbazine, methotrexate, 5-fluorouracil, prednisone, tamoxifen); CMF methotrexate); CPOB (cyclophosphamide, prednisone, PTH (cyclophosphamide, methotrexate, 5-fluorouracil, Oncovin, bleomycin); CPT-11 (camptothecin-11); CPTR prednisone, tamoxifen, Halotestin); CMFP-VA (cyproterone); CRA (cis-retinoic acid); 13-CRA (13-cis (cyclophosphamide, methotrexate, 5-fluorouracil, retinoic acid); Crasnitin (C-asparaginase); CRF-187; crisna prednisone, Vincristine, Adriamycin); CMFT 40 tol mesylate; CROP (cyclophosphamide, rubidazone, (cyclophosphamide, methotrexate, 5-fluorouracil, Oncovin, prednisone); CROPAM (cyclophosphamide, tamoxifen); CMF-TAM (cyclophosphamide, methotrexate, rubidaZone, Oncovin, prednisone, L-asparaginase, 5-fluorouracil, tamoxifen); CM-5-FU (cyclophosphamide, methotrexate); CT (cytarabine, thioguanine); CTCb methotrexate, 5-fluorouracil); CMFV (cyclophosphamide, (cyclophosphamide, , carboplatin); CTX methotrexate, 5-fluorouracil, Vincristine); CMFVAT 45 (cyclophosphamide); CTX-Plat (cyclophosphamide, (cyclophosphamide, methotrexate, 5- fluorouracil, Platinol); CV 205-502; CV (cisplatin, VP-16); CVA Vincristine, Adriamycin,; testosterone); CMFVP (cyclophosphamide, Vincristine, Adriamycin); CVA-BMP (cyclophosphamide, methotrexate, 5-fluorouracil, (cyclophosphamide, Vincristine, Adriamycin, BCNU, Vincristine, prednisone); CMH (cyclophosphamide, methotrexate, ; procarbazine); C-VAD, CVAD m-AMSA, hydroxyurea); CMOPP, C-MOPP 50 (cyclophosphamide, Vincristine, Adriamycin, (cyclophosphamide, mechlorethamine, Onco Vin, dexamethasone); CVB (CCNU, vinblastine, bleomycin); procarbazine, prednisone); CMP (CCNU, methotrexate, CVBD (CCNU, vinblastine, bleomycin, dexamethasone); procarbazine); CMPF (cyclophosphamide, methotrexate, CVD (cisplatin, vinblastine, dacarbazine); CVEB (cisplatin, prednisone, 5-fluorouracil); CMV (cisplatin, methotrexate, Velban, etoposide, bleomycin); CVI (carboplatin, VePesid, vinblastine); CNF (cyclophosphamide, Novantrone, 55 ifosfamide, Mesne X uroprotection); CVM 5-fluorouracil); CNOP, C-NOP (cyclophosphamide, (cyclophosphamide, Vincristine, methotrexate); CVP No vant rone, Onco vin, prednisone); CO AP (cyclophosphamide, Vincristine, prednisone); CVP-BLEO (cyclophosphamide, Oncovin, ara-C, prednisone); COAP (cyclophosphamide, Vincristine, prednisone, bleomycin); BLEO (cyclophosphamide, Oncovin, ara-C, prednisone, CVPP (CCNU, vinblastine, prednisone, procarbazine); bleomaycin); COB (cisplatin, Oncovin, bleomycm); COB 60 CVPP (cyclophosphamide, vinblastine, procarbazine, MAM (cyclophosphamide, Oncovin, bleomycin, prednisone); CVPP-CCNU (cyclophosphamide, vinblastine, methotrexate, Adriamycin,; MeCCNU); COF/COM procarbazine, prednisone, CCNU); CY (cyclophosphamide (cyclophosphamide, Onco Vin, 5-fluorou racil/ or Cytoxan); CyADIC (cyclophosphamide, Adriamycin, cyclophosphamide, Oncovin,; methotrexate); colaspase, DTIC); CYC (cyclophosphamide); cyclocreatine; cyclo COM (cyclophosphamide, Oncovin, methotrexate); COM 65 hexylnitrosourea (CCNU); cyclophosphamide (CPA or (cyclophosphamide, Oncovin, MeCCNU); COMA-A CPM or CYC); Cycoblastin (cyclophosphamide); CyHOP (cyclophosphamide, Oncovin, methotrexate, Adriamycin, (cyclophosphamide, Halotestin, Oncovin, prednisone); US 6,319,682 B1 27 28 cyproterone (CPTR); cysteamine dichloroplatinum2; CYT Cooperative Oncology Group (ECOG); EBAP (Eldisine, 103-Y-90 (OncoRad); CytaBOM, CYTABOM (cytarabine, BCNU, Adriamycin, prednisone); echinomycin; EBRT bleomycin, Oncovin, mechlorethamine); Cytadren (external beam radiation therapy); ECHO (etoposide, (aminoglutethimide); cytarabine (ara-C or Cytosar-U or cyclophosphamide, hydroxy daunomycin, Oncovin); cytosine arabinoside); Cytosar-U (cytarabine); cytosine ara 10-EDAM (10-ethyl-10-deaza-aminopterin) (Edatrexate); binoside (ara-C); Cytoxan (CTX); Cytoxan Lyophilized; ECMV (etoposide, Cytoxan, methotrexate, Vincristine); Cy VAD ACT, CY-VA-DACT (cyclophosphamide, ECOG (Eastern Cooperative Oncology Group); EDAM (10 Vincristine, Adriamycin, dactinomycin); CyVADIC ethyl-deaza-aminopterin or 10-EdAM); EDAP (etoposide, (cyclophosphamide, Vincristine, methotrexate, Adriamycin, dexamethasone, ara-C, Platinol); Edatrexate (10-EDAM) DTIC); Cy VMAD (cyclophosphamide, Vincristine, (10-ethyl-10-deaza-aminopterin); efornithine (DFMO); methotrexate, Adriamycin, DTIC); DA (daunorubicin, ara Efudex (fluorouracil); EFP (etoposide, fluorouracil, C), dacarbazine (DTIC); DACT (dactinomycin); dactino Platinol); Einhorn regimen; Eldisine ( sulfate); mycin (actinomycin D); DAP (dianhydrogalactitol, ELF (etoposide, leucovorin, 5-fluorouracil); Elipten Adriamycin, Platinol); DAP/TMP (dapsone, trimethoprim); (aminoglutethimide); Eliott's B Solution; ellipticine; DAT (daunomycin, ara-C, 6-thioguanine); DATVP 15 ; Elspar (asparaginase); EMACO (etoposide, (daunomycin, ara-C, 6-thioguanine, Vincristine, methotrexate, actinomycin D, cyclophosphamide, Oncovin); prednisone); Datelliptium (ellipticine), daunomycin Emcyt (estramustine); E-MVAC (escalated methotrexate, (DNM); daunorubicin hydrochloride (Cerubidine or DNR); vinblastine, Adriamycin, cyclophosphamide); Endoxan DaunoXome (liposomal daunorubicin); DAV Asta (cyclophosphamide); enloplatin; enpromate; EP (dibromodulcitol, Adriamycin, Vincristine); DAVA (etoposide, Platinol); EPI (epirubicin); epi-ADR (desacetyl vinblastine amide); DAVH (dibromodulcitol, (epinephrine-Adriamycin); epipropidine, epirubicin; epiru Adriamycin, V in cristine, Halo testin); D BD bicin HCl; EPO (erythropoietin); EPOCH (etoposide, (dibromodulcitol); DBV (dacarbazine, BCNU, Vincristine); prednisone, Oncovin, cyclophosphamide, Halotestin); Epo DC (daunorubicin, cytarabine); DCCMP (daunorubicin, dyl, Epogen; Eprex (erythropoietin), erbulozole; Ergamisol cyclocytidine, 6-metacaptopurine, prednisone); DCF 25 (levamisole); Erwinase; erwinia asparaginase; erwinia (2-deoxyco for mycin or pen to statin); DCM L-asparaginase; ESHAP (etoposide, Solu-Medrol, ara-C, (dichloromethotrexate); DCMP (daunorubicin, cytarabine, Platinol); esorubicin; esorubicin HCl; esperamycin; Esteri 6-metacaptopurine, prednisone); DCT (daunorubicin, fied estrogens, Estinyl; Estra-L (estradiol); Estrace; estra cytarabine, thioguanine); DCV (dacarbazine, CCNU, diol, Estradurin; estramustine (Emcyt); Estratab, Estraval; Vincristine); DDP (cis-diaminodichloroplatinum or Estrone 5; etanidazole; ethacrynic acid; ethinyl estradiol; cisplatin); 3-deazaguanine; DECAL (dexamethasone, Ethiodol; Ethiofos (WR2721); 10-ethyl-10-deaza etoposide, cisplatin, ara-C, L-asparaginase); Decapeptyl aminopterin (10-EDAM) (Edatrexate); Ethyol (amifostine); (triptorelin); ; deferoxamine; Deladiol-40 etoposide (VP-16, VePesid); etoposide phosphate; etoprine; (estradiol); Delatest (testosterone); Delatestry1 Eulexin; EVA (etoposide, vinblastine, Adriamycin); EVAP; (testosterone); Delestrogen (estradiol); 2-deoxy-5- 35 Everone; EVMAC (escalated methotrexate, vinblastine, azacitidine (DAC); deoxycoformycin (DCF or ); Adriamycin, cisplatin); FAC (5-fluorouracil, Adriamycin, deoxyspergualin (DSG); depandro, depAndro 200 cyclophosphamide); FAC-BCG (Ftorafur, Adriamycin, (testosterone); Depofoam encapsulated cytarabine (DTC cyclophosphamide, BCG); FAC-LEV (5-fluorouracil, 101); Depo-Provera; Depotest (testosterone); Depo Adriamycin, cyclophosphamide, levamisole); FAC-M Testosterone; deXormaplatin; deXVerapamil, deZaguanine; 40 (5-fluorouracil, Adriamycin, cyclophosphamide, dezaguanine mesylate; DFDC (); DFMO methotrexate); FACP (Ftorah fur, Adriamycin, (eflornithine); DFMO-MGBG; DFV (DDP, 5-fluorouracil, cyclophosphamide, Platinol); FACS (5-fluorouracil, VePesid); DHAC (azacitidine); DHAD (mitoxantrone); Adriamycin, cyclophosphamide, Streptozocin); FACVP DHAP (dexamethasone, high-dose ara-C, Platinol); DI 694; (5-fluorouracil, Adriamycin, cyclophosphamide, VP-16); diaziquone (aziridinylbenzoquinone); dibromodulcitol 45 fadrozole; fadrozole HCl (Arensin); FAM (5-fluorouracil, (Mitolactol); Dicorvin; didemnin B; didox; diethyldithiocar Adriamycin, mitomycin-C); FAM-C (5-fluorouracil, bamate (DTC); diethylstilbestrol diphosphate; dihematopor Adriamycin, methyl-CCNU); FAM-CF (5-fluorouracil, phyrin ethers; dihydro-5-azacitidine (azacitidine or DHAC); Adriamycin, mitomycin, citrovorum factor); FAME, FAMe DIMOPP (dose-intensified MOPP); Dioval XX, Dioval 40 (5-fluorouracil, Adriamycin, MeCCNU); FAMMe (estradiol); DL (doxorubicin, lomustine); DM 50 (5-fluorouracil, Adriamycin, mitomycin-C, MeCCNU); (dexamethasone); DMC (dactinomycin, methotrexate, FAMP ( monophosphate); FAM-S cyclophosphamide); DMP 840; DNM (daunomycin); DNR (5-fluorouracil, Adriamycin, mitomycin-C, Streptozocin); (daunorubicin); DOAP (daunorubicin, Oncovin, ara-C, FAMTX (5-fluorouracil, Adriamycin, highdose prednisone); , dolasestron; DOX (doxorubicin); methotrexate); FAP (5-fluorouracil, Adriamycin, Platinol); Doxil (liposome formulation of doxorubicin); doxorubicin 55 Farmorubicin (epirubicin HCl); fazarabine (ara-AC); FCAP (Adriamycin or DOX); Drolban; droloxifene; dromo (5-fluorouracil, cyclophosphamide, Adriamycin, Platinol); Stanolone propionate (Drostanolone); dronabino1, FCE (5-fluorouracil, epirubicin, cyclophosphamide); F-CL Drostanolone; DTC (diethyldithiocarbamate); DTIC (5-fluorouracil, leucovorin calcium); FCP (5-fluorouracil, (dacarbazine); DTIC-ACTD (DTIC, actinomycin D); DTIC cyclophosphamide, prednisone); FEC (5-fluorouracil, Dome (dacarbazine); duaZomycin; Durabolin (nandrolone 60 epirubicin, cyclophosphamide); FED (5-fluorouracil, phen propionate); Duragen (estradiol); Duralutin etoposide, DDP); Feminone (discontinued in 1993) (ethyl (hydroxyprogesterone); Duratest (testosterone); Durathate estradiol); fenretinide (4-HPR); filgrastim (Neupogen) (testosterone); DVB (DDP, vindesine, bleomycin); DVP (G-CSF); FIMe (5-fluorouracil ICRF-159, MeCCNU); fin (daunorubicin, Vincristine, prednisone); DVPL-ASP aste ride (MK-906); 5+2 protocol (cytarabine, (daunorubicin, Vincristine, prednisone, L-asparaginase); 65 daunorubicin); FK506; FL (flutamide, leuprolide acetate); DZAPO (daunorubicin, azacitidine, ara-C, prednisone, FL (flutamide, Lupron depot); FLAC (5-fluorouracil, leuco Oncovin); EAP (etoposide, Adriamycin, Platinol); Eastern Vorin calcium, Adriamycin, cyclophosphamide); FLAG-Ida; US 6,319,682 B1 29 30 FLAP (5-fluorouracil, leucovorin, Adriamycin, Platinol); human IgM monoclonal antibody to cytomegalovirus; HXM FLe (5-fluorouracil, levamisole); (FUdR); flucy (hexamethylmelamine); Hybolin Improved (nandrolone tosine (Ancobon or 5-FC); Fludara (fludarabine); fludara phenpropionate); Hybri-CEAker; Hy-Gestrone; hydrazine bine (FAMP or Fludara); fludarabine phosphate; fluorode Sulfate, 4-hydroxyperoxycyclophosphamide; Hydrea oxyuridine (FUdR); Fluoroplex cream, topical (hydroxyurea), 4-hydroperoxycyclophosphamide (4-HC); (fluorouracil); fluorouracil (Adrucil or Efudex or ; hydroxyurea (Hydrea); Hylutin; 5-fluorouracil or 5-FU or Fluoroplex); Fluosol/BCNU Hyprogest; Hyproval; Hyproxon; ICE (ifosfamide, (Fluosol-DA20, BCNU); fluoxymesterone (FMX or carboplatin, etoposide); ICI D1694 (Tomudex); ICRF-187; Halotestin); fluorodeoxyuridine (FUDR); Fluoroplex IDA (); Idamycin (idarubicin HCl); idaribicin; (5-fluorouracil); flurocitabine; flutamide (Eulexin or FLUT); idarubicin HCl; IDEC-C2B8; IDMTX (intermediate-dose FMS (5-fluorouracil, mitomycin-C, streptozocin); FMV methotrexate); IDX (4'-iodo-4'deoxydoxorubicin); Ifex (5-fluorouracil, methyl CCNU, vincristine); FNM (ifosfamide); IFF (ifosfamide); IFLrA (recombinant inter (5-fluorouracil, Novantrone, methotrexate); FOAM feron alpha); IFM (ifosfamide); IFN-A (interferon alpha); (5-fluorouracil, Oncovin, Adriamycin, mitomycin-C); Folex IFN-alpha 2a (interferon alpha 2a); IFN-G (gamma (mnethotrexate) (discontinued in 1992); Folex PFS 15 interferon); IFOS (ifosfamide); ifosfamide; IFX (methotrexate); folinic acid (leucovorin); FOM (ifosfamide); IL-2 (interleukin-2); ilmofosine; IMF (Ifex, (5-fluorouracil, Oncovin, mitomycin-C); FOMi mesa, Folex, 5-fluorouracil); imidazole carboxamide (5-fluorouracil, Oncovin, mitomycin-C); Fosfestrol (dacarbazine); ImmTher; ImmuRAID-AFP; ImmuRAID (diethylstilbestrol diphosphate); fosquidone; fostriecin; fos CEA, ImmuRAID-hCG, ImmuRAID-LL2, ImmuRAIT triecin sodium, (S 10036); FRACON LL2, Imuran (azathioprine), Imuvert (Serratia marcescens (framycetin, collistin, nyStatin); F to rafur (1,2- extract); IMVP-16 (ifosfamide, methotrexate, VP-16); inter tetrahydrofuranyl-5-fluorouracil); 5-FU (5-fluorouracil); feron alfa, interferon alfa-2a, interferon alfa-2b; interferon FUDR (floxu ridine); FUdR (5-fluorou racil beta (Serono, R-Frone); interferon gamma-1b; interleukin-1, deoxyribonucleoside); FUM (5-fluorouracil, methotrexate); 1a, 2.3, 4, 6, 11; intoplicine; lintron-A, iododeoxyuridine, Fungizone (amphotericin-B); FUra (fluorouracil); FURAM 25 isotretinoin; iododeoxyuridine; IPP (isopropyl pyrrolizine); (Ftorafur, Adriamycin, mitomycin-C); Furhman nuclear iproplatin; iridium; irimotecan; Isorvorin (L-leucovorin); IT grade; FUVAC (5-fluorouracil, vinblastine, Adriamycin, MTX (intrathecal methotrexate); IUdR (idoxuridine); cyclophosphamide); FXM (fluoxymesterone or Halotestin); JT1001; Kestrone 5; KGC (Keflin, gentamicin, FZ (flutamide, Zoladex); galamustine (G-6-M); gallium carbenicillin); Kyna cyte; ladakamycin; L. A.E. 20 nitrate (GAN); Gamimune Normal (immune globulin); gem (discontinued in 1992); L AM (L-asparaginase, citabine (DFDC) (Gemzar); gemcitabine HCl; Gemzar methotrexate); lanre otide acetate; LAPOCA (gemcitabine); Gesterol (progesterone); GL331; Gliadel; (L-asparaginase, prednisone, Oncovin, cytarabine, glycyrrhetinic acid; GM-CFU (granulocyte-macrophage Adriamycin); L-ASP (L-asparaginase); L-aspaaginase; colony-forming unit); GM-CSF (granulocyte-macrophage L-buthionine sulfoximine; L-CF (leucovorin-citrovorum colony-stimulating factor); GOD-26MM; gold Au 198; gos 35 factor); LCR (Vincristine); LDI-200; letrozole; Leucomax erelin acetate (ZDX or Zoladex); GVAX; Gynogen L.A. (molgramoStim); L-leucovorin; leucovorin calcium; Leuke “10", Gynogen L.A. "20", Gynogen L.A. “40” (estradiol); ran (chlorambucil); Leukine (SargramoStim); Leukoglobu H-447; H (Halotestin); HAD (hexamethylenamine, lin; leuprolide acetate (Leuprorelin); Leuprorelin; leurocris Adriamycin, DDP); Halo drin; Halotestin; HAM tine; Leustatin (2-CdA or cladribine); LEV (levamisole (hexamethylenamine, Adriamycin, melphalan); HAM 40 hydrochloride); levamisole (Ergamisol); LGD-1069; liaro (hexamethylenamine, Adriamycin, methotrexate); HAMP Zole fumarate; liarozole HCl, linker protocol; Linomide (hexamethylenamie, Adriamycin, methotrexate, Platinol); (roquinimex), Lipidox (liposomal doxorubicin); liposomal HC (hydrocortisone); HCAO (hexamethylenamine, doxorubicin (TLCD-99); liposomal MT-PE; liposomal nys cyclophosphamide, Adriamycin, Platinol); H-CAP tatin; L-leucovorin (Isovorin); LMF (Leukeran, (hexamethylenamine, cyclophosphamide, Adriamycin, 45 methotrexate, 5-fluorouracil); L-PAM (L-phenylaline mus Platinol), 4-HC (4-hydroperoxycyclophosphamide or tard or melphalan); lobaplatin; LOMAC (leucovorin, Pergamid); HDARA-C (high-dose ara-C); HDC Oncovin, methotrexate, Adriamycin, cyclophosphamide); (pentostatin); HDMTX (high-dose methotrexate); HDMTX lometrexol; lomustine (CCNU); lonidamine; ; CF (high-dose methotrexate, citrovorum factor); HDMTX/ losoxantrone HCl; LP 2307; L-PAM (L-phenylalanine LV (high-dose methotrexate, leucovorin); HDPEB (high 50 mustard) (melphalan); Lupron; Lupron Depot; LuVax dose PEB or Platinol, etoposide, bleomycin); HD-VAC (monoclonal antibodies); LV (leucovorin); L-VAM (Lupron, (high-dose methotrexate plus vinblastine, Adriamycin, Velban, Adriamycin, Mutamycin); LVVP (Leukeran, cisplatin); hepsulfam, HER2 (monoclonal antibody); Hexa vinblastine, Vincristine, prednisone); Lysodren; M2 CAF (Hexalen, cyclophosphamide, Adrucil, Folex); (Vincristine, carmustine, cyclophosphamide, melphalan, Hexalen (altretamine); hexamethylene; hexamethylene 55 prednisone); MAb, MAB (monoclonal antibody); MAb bisacetamide; hexamethylmelamine, Hexastat (altretamine); B72,3; MAb-L6; MABOP (Mustargen, Adriamycin, HiC-COM (ara-C, citrovorum factor, allopurinol, Elliot B bleomycin, Oncovin, prednisone); MAC (methotrexate, Solution, cyclophosphamide, Oncovin, methotrexate); actinomycin D, cyclophosphamide); MAC (methotrexate, HIDAC, HilDAC (high-dose ara-C); high-risk ATAC Adriamycin, cyclophosphamide); MAC (mitomycin-C, (L-asparaginase, ara-C, VP-16, anti-J2 26 monoclonal 60 Adriamycin, cyclophosphamide); MACC (methotrexate, antibody, anti-CALLA hybridoma antibody); Histerone; Adriamycin, cyclophosphamide, CCNU); MACHO HMBA (hexamethylene bisacetamide); HMM (Hexalen); (methotrexate, asparaginase, cyclophosphamide, HMTX (high-dose methotrexate); HN2 (nitrogen mustard); hydroxydaunomycin, Oncovin); MACOP-B (methotrexate, HOAP-BLEO (hydroxydaunomycin, Oncovin, ara-C, Adriamycin, cyclophosphamide, Oncovin, prednisone, prednisone, bleomycin); homoharringtonine; HOP 65 bleomycm); Macrolin (mnacrophage colony-stimulating (hydroxy daunomycin, Oncovin, prednisone); HPR factor); MacStim (macrophage colony-stimulating factor); (fenretinide); 4-HPR (fenretinide); HU (hydroxyurea); MAD (MeCCNU, Adriamycin); MADDOC US 6,319,682 B1 31 32 (mechlorethamine, Adriamycin, dacarbazine, DDP, Adriamycin); Modrastane; MOF (MeCCNU, Oncovin, Oncovin, cyclophosphamide); MAID (mesna, Adriamycin, 5-fluorouracil); MOF (methotrexate, Oncovin, interleukin-3, dacarbazine); MAID (Mesnex, Adriamycin, 5-fluorouracil); MOF-STREP (MeCCNU, Oncovin, Ifex, dacarbazine); maitansine; MAK 195 F (monoclonal 5-fluorouracil); molgramostim; MOMP (mechlorethamine, antibodies); m-AMSA (amsacrine); MAP (melphalan, Oncovin, methotrexate, prednisone); monoclonal antibod Adriamycin, prednisone); MAP (mitomycin-C, Adriamycin, ies, monoclonal antibodies recognizing B-cell Platinol); masoprocol; MAT(multiple agent therapy); Matu idiotypes, monoclonal antibody 17-1A, monoclonal anti lane (procarbazine); maytansine; MAZE (m-AMSA, body PM-81; monoclonal antibody PM-81 & AML2-23; azacitidine, etoposide); M-BACOD (high-dose Monogen; monomercaptoundecahydro-cloSO-DQ decabo methotrexate, bleomycin, Adriamycin, cyclophosphamide, rate sodium; Monox-IX; MOP (mechlorethamine, Oncovin, Oncovin, dexamethasone); M-BACOS (methotrexate, prednisone); MOP (mechlorethamine, Oncovin, bleomycin, Adriamycin, cyclophosphamide, Oncovin,; procarbazine); MOP (melp halan, Onco Vin, Solu-Medrol); M-BAM (cyclophosphamide, total body methylprednisolone); MOP-BAP (mechlorethamine, irradiation, monoclonal antibodies); MBC (methotrexate, Oncovin, procarbazine, bleomycin, Adriamycin, bleomycin, cisplatin); MBD (methotrexate, bleomycin, 15 prednisone); MOPP (mechlorethamine, Oncovin, cisplatin); MC (mitoxantrone, cytarabine); MCBP procarbazine, prednisone); MOPP (methotrexate, Oncovin, (melphalan, cyclophosphamide, BCNU, prednisone); MCP procarbazine, prednisone); MOPP-ABV (mechlorethamine, (melphalan, cyclophosphamide, prednisone); M-CSF Oncovin, procarbazine, prednisone, Adriamycin,; (macrophage colony-Stimulating factor); MCV bleomycin, vinblastine); MOPP-ABVD (mechlorethamine, (methotrexate, cisplatin, vinblastine); MD 28314OA; Oncovin, procarbazine, prednisone, Adriamycin,; MDAM; MDL18, 962; MDL 73,147EF; MDLO bleomycin, vinblastine, dacarbazine); MOPP-LO BLEO (me to clopramide, de Xame thaSone, lo razepam, (mechlorethamine, Oncovin, procarbazine, prednisone, ondansetron); MDX210 (humanized anti-cancer bispecific bleomycin); MOPPHDB (mechlorethamine, Oncovin, antibody); MeCCNU (methyl-CCNU or ); procarbazine, prednisone, high-dose bleomycin), MOP me chlore thamine; ME CY (methotrexate, 25 PLDB (mechlorethamine, Oncovin, procarbazine, cyclophosphamide); medroxyprogesterone; MeFA (methyl prednisone, low-dose bleomycin); MOPr (meclorethamine, CCNU, 5-fluorouracil, Adriamycin); Megace (megestrol Oncovin, procarbazine); MP (melphalan, prednisone); 6-MP acetate); megestrol acetate (Megace); Melacine (melanoma (6-mercaptopurine); MPFL (methotrexate, Platinol, vaccine); melanoma vaccine (Melacine); melanoma cell 5-fluorouracil, leucovorin, calcium); MPL (melphalan); lysate vaccine; melengestrol acetate; MeliBu (buSulfan); MPL+PRED (melp halan, prednisone); M TX Melimmune-1 (monoclonal antibodies); Melimmune-2 (methotrexate); MTX+MP (methotrexate, mercaptopurine); (monoclonal antibodies); melphalan; melphalan IV; MelVax MTX+MP+CTX (methotrexate, mercaptopurine, Cytoxan); (monoclonal antibodies); Memorial Sloan-Kettering proto Murine L6 monoclonal antibody; Mu Starge n col; Menest (esterified estrogens); menogaril; MePRDL (mechlorethamine HCl); mustine HCl; Mutamycin (methylprednisolone); merbarone; mercaptopurine; 35 (mitomycin); MV (mitoxantrone, VP-16); MVAC 6-mercaptopurine (6-MP or Purinethol); mesna (Mesnex); (methotrexate, vinblastine, Adriamycin, cisplatin); MVF MesneX (mesna); methionyl granulocyte CSF, recombinant; (mitoxantrone, Vincristine, 5-fluorouracil); MVP methionyl human granulocyte CSF, recombinant; Metho (mitomycin-C, vinblastine, Platinol); MVPP sarb; methotrexate (MTX); methotrexate LFP sodium, meth (mechlorethamine, vinblastine, procarbazine, prednisone); otrexate Sodium; methotrexate/AZone (methotrexate, 40 MVT (mitoxantrone, VP-16, thiotepa); MVVPP laurocapram); methyl-CCNU (semustine or MeCCNU); (mechlorethamine, Vincristine, vinblastine, procarbazine, methylmercaptopurine; Methylprednisolone; Meticorten; prednisone); Myleran; Mylosar (5-azacytidine); N-901 metoprine; Mexate; MF (methotrexate, 5-fluorouracil); MF blocked ricin (Oncolysin S); NAC (nitrogen mustard, (mitomycin, 5-fluorouracil); MFP (melphalan, Adriamycin, CCNU); Nadrobolic (discontinued in 1994); 5-fluorouracil, Provera); MGDF (megakaryocyte growth 45 nafidimide (amonafide); Navelbine (); neon par and development factor); MICE (mesna rescue, ifosfamide, ticle; Neosar (cyclophosphamide); neptamustine; NEU dif carboplatin, etoposide); MIFA (mitomycin, fluorouracil, ferentiation factor; Neupogen (filgrastim); Neu-Sensamide; Adriamycin); mifepristone (RU486); MIH (procarbazine); N-methylhydrazine, nimustine; Nipent (pentostatin), nitro MINE (mesna, ifosfamide, Novantrone, etoposide); MINE/ gen mustard; NM (mechlorethamine); N-methylhydrazine ESHAP; mini-COAP (cyclophosphamide, Oncovin, ara-C, 50 (procarbazine); N-monomethyl-L-arginine (MNA), nocoda prednisone); MINT, MIP-1 alpha; misonidazole; Mithracin Zole; nogalamycin; Nolvadex (tamoxifen); Novantrone (); mithramycin (now plicamycin), mitindomide; (mitoxantrone); NovoSeven; Numega (interleukin-11); OAP MITO (mitomycin-C); mitocarcin; mitocromin; mitogillin; (Oncovin, ara-C, prednisone); OAP-BLEO (Oncovin, ara-C, , mitolactol (dibromodulcitol), mitomalcin, prednisone, bleomycin); OctreoScan 111 (Indium In 111 mitomycin; mitomycin-C (MMC or Mutamycin); mitosper; 55 pentetreotide); octreotide (Sandostatin); O-DAP (Oncovin, mitot a ne; mitoxantrone (No vant rone); MM dianhydrogalactitol, Adriamycin, Platinol); OLX-102; (mercaptopurine, methotrexate); MMC (mitomycin-C); OMAD (Oncovin, methotrexate, Adriamycin, MMOPP (methotrexate, mechlorethamine, Oncovin, dactinomycin), onapristone; Oncaspar (); pro carb a Zine, prednis one); MMPR Oncoject; Oncol; Oncolym; Oncolysin B, Oncolysin S; (methylmercaptopurine); MMPT (methylprednisolone pulse 60 Onco Non-Small Cell; OncoPurge; OncoRad OV103 (CYT therapy); MNA (N-monomethyl-L-argmine); MOAB, 103-Y-90); OncoRad Bladder; OncoRad Ovarian; OncoRad Mo Ab , MAB (monoclonal antibody); MOAD Prostate; Onco Scint CR/OV: Onco Scint OV103 (methotrexate, Oncovin, L-asparaginase, dexamethasone); (celogobab); OncoScint PR; OncoTrac; Oncovin MOAP (PEG-asparaginase, Oncovin, methotrexate, (Vincristine); Oncozole (3-deazaguanine); ondansetron; prednisone); MOB (Mustargen, Oncovin, bleomycin); 65 OPAL (Oncovin, prednisone, L-asparaginase); OPEN MOB-III (mitomycin-C, Oncovin, bleomycin, cisplatin); (Oncovin, prednisone, etoposide, Novantrone); OPP MOCA (methotrexate, Oncovin, cyclophosphamide, (Oncovin, procarbazine, predisone); OPPA (Oncovin, US 6,319,682 B1 33 34 procarbazine, prednisone, Adriamycim); Ora-Testryl, orma (adoptive cellular therapy); riboprine; ricin (blocked) con platin; ornithine; Orthozyme-CD5; Ossirene; ovarian jugated murine monoclonal antibody; RIDD (recombinant rhenium-186 MAb; oxisuran; P32; PAB-Esc-C (Platinol, interleukin-2, dacarbazine, DDP); RMP-7; ROAP Adriamycin, ble o mycin, e Scalating doses of (rubidazone, Oncovin, ara-C, prednisone); Roferon-A; cyclophosphamide); PAC (Platinol Adriamycin, Rogletimide (pyridoglutethimide); Rubrex (doxorubicin); cyclophosphamide); PACE (Platinol, Adriamycin, rufocromomycin; St. Jude Research Children's Hospital cyclophosphamide, etoposide); (Taxol); PALA protocol; Salagen; SAM (streptozocin, Adriamycin, methyl (N-phosphonoacetyl-L-asparate); PAM, L-PAM CCNU); Sandimmune (cyclosporine); Sandostatin (phenylalanine mustard); Panel Quest; Panorex (monoclonal (octreotide); SargramoStim (Prokine), SCAB (streptozocin, antibody 17-1A); Paraplatin (carboplatin); PATCO CCNU, Adriamycin, bleomycin); semustine (methyl (prednisone, ara-C, thioguanine, cyclophosphamide, CCNU); Serono (interferon beta, R-Frone); Serratia marce Oncovin); PAVe (procarbazine, Alkeran, Velban); PBV Scens extract; 7+3 protocol (cytarabine, daunorubicin); (Platinol, bleomycin, vinblastine); PCB (procarbazine); PCE 7U85; Sialyl Tn-KLH; SIMAL-pilot (ara-C, hydrocortisone, (Platinol, cyclophosphamide, Eldisine); PCNU; PCV mesna, prednisone, VP-16, leucovorin); SIMAL-second (procarbazine, CCNU, Vincristine); PEB (Platinol, 15 induction/maintenance (prednisone, L-asparaginase, etoposide, bleomycin); PEC (Platinol, etoposide, daunomycin, VM-26,; methotrexate, ara-C, VP-16, cyclophosphamide); PEG-ADA (Adagen, pegademase leucovorin); SIMAL-bone marrow transplant (ara-C, bovine); PEG-ASP (PEG-asparaginase); PEG-L- methotrexate, prednisone); Simon capsule (cervical); asparaginase; pegaspargase; peliomycin, Penclomedine, SimtraZene, Single-chain antigen-binding proteins, SK pent a mustine; pen to statin; PEP (Procytox, (Sloan-Kettering protocol, SMF (Streptozocin, mitomycin epipodophyllotoxin-derivative, prednisolone); peplomycin; C, 5-fluorouracil); Sparfosic acid, SparSomycin; Specifid perfosfamide; Pergamid; PF+E (Platinol, 5-fluorouracil plus (discontinued in 1994); Spherex, Spirogermanium; Spiro etoposide, methotrexate, leucovorin); PFL (Platinum, mustine; spiroplatin; S-P-T “liquid'capsules; SR-2508; 5-fluorouracil, leucovorin calcium); PFM (Platinol, SSTN (octreotide or Sandostatin); ST1-RTA immunotoxin; 5-fluorouracil, methotrexate); PFT (phenylalanine mustard, 25 STAMP protocol (Solid Tumor Autologous Bone Marrow 5-fluorouracil, tamoxifen), phenylalanine mustard; phenyl Program); STEAM (Streptonigrin, thioguanine, butyrate; Phosphocol P32; Photofrin; PHRT (procarbazine, cyclophosphamide, actinomycin, mitomycin); STEPA hydroxyurea, radiotherapy); PIA (Platinol, ifosfamide, (thiotepa); Sterecyt; Stilphostrol; Streptonigrin, Streptozo Adriamycin); ; piposulfan, ; piraZO cin; , SU101; Sulofenur, Super-CM regimen fuirin; piritrexim; piroxantrone; PIXY; PIXY 321; Pixykine; (cyclophosphamide, methotrexate, 5-fluorouracil); Suprefact Platinol (cisplatin); Platinol-AQ, platinum diamminodichlo (buserelin); Suramin; suramim sodium; SWOG CMFVP ride; plicamycin; plomestane; PM (prednimustine); PMB (cyclophosphamide, methotrexate, 5-fluorouracil, (Platinol, methotrexate, bleomycin); PMF (phenylalanine Vincristine, prednisone); synercid; T-2 protocol mustard, methotrexate, 5-fluorouracil); PMFAC (dactinomycin, Adriamycin, Vincristine, cyclophosphamide, (prednisone, methotrexate, 5-fluorouracil, Adriamycin, 35 radiation); T-10 protocol (methotrexate, calcium leucovorin cyclophosphamide); P-MVAC (Platinol, methotrexate, rescue, Adriamycin, cisplatin, ble o my cin, vinblastine, Adriamycin, carboplatin); POC (procarbazine, cyclophosphamide, dactinomycin); T4N5 (T4 endolase V, Oncovin, CCNU); POCA (prednisone, Oncovin, cytarabine, liposone encapsulated); Tace (chlorotrianisene); TAD Adriamycin); POCC (pro carbazine, Oncovin, (6-thioguanine, ara-C, daunomycin); talisomycin; tallimus cyclophosphamide, CCNU); POG protocol (Pediatric 40 tine; TAM (tamoxifen); tamoxifen; Tarabine PFS Oncology Group); polyestradiol; POMP (prednisone, (cytarabine); Tasmar; tauromustine; Taxol (paclitaxel); Oncovin, methotrexate, Purinethol); ; por Taxotere (docetaxel); TBC-CEA, TBI (total body firomycin; porton asparaginase; PPI-002; PRDL irradiation); TC (6-thioguanine, cytarabine); T-CAP (Baker (prednisolone); PRED (prednisone); prednimustine; PRH-E Antifol, cyclophosphamide, Adriamycin, Platinol); T-CAP (Platinol, etanidazole); PRIME (procarbazine, ifosfamide, 45 III (triazinate, cyclophosphamide, Adriamycin, Platinol); methotrexate); Pro-Depo; procarbazine; Procrit; Progens, TCN-P (triciribine phosphate); TEC (thiotepa, etoposide, Progynon pellets; Prokine (SargramoStim); Proleukin carboplatin); teceleukin; teleleukin; teloxantrone; temozo (aldesleukin); Proloid (discontinued in 1992); ProMACE lomide; TEMP (tamoxifen, etoposide, mitoxantrone, (prednisone, methotrexate, Adriamycin, cyclophosphamide, Platinol); teniposide (VeeM-26); terephthalamidine; terox etoposide); ProMACE-Cyta BOM (prednisone, 50 irone; Teslac (); TESPA (thiotepa); Testamone methotrexate, Adriamycin, cyclophosphamide, etoposide, (testosterone); Testaqua, Testex (testosterone); testolactone; cytarabine, bleomycin, Oncovin, methotrexate); ProMACE Testone (testosterone); Testred (testosterone); Testrin MOPP (procarbazine, methotrexate, Adriamycin, (testosterone); Testroject (testosterone); IFN tumor factor cyclophosphamide, etoposide, MuStargen, Oncovin, necrosis; Theelin Aqueous (estrone); TheraCys (BCG procarbazine, prednisone); properomine protocol; ProScar; 55 vaccine); Theradex (AD-32); TheraSeed; thiamiprine; pulse VAC (Vincristine, actinomycin D, cyclophosphamide); thioguanine (Lanvis or 6-TG or 6thioguanine); Purinethol (mercaptopurine); puromycin; PUVA (psoralens, 6-thioguanine (6-TG or thioguanine), 6-TG (6-thioguanine); ultraViolet A); PVA (prednisone, Vincristine, asparaginase); thiosulfate; thiotepa (STEPA or TESPA or TTPA); thymi PVB (Platinol, vinblastine, bleomycin); PVDA (prednisone, dine; Thymosin Alpha-I; thymidine; Thyroid Strong; tiami Vincristine, daunorubicin, asparaginase); PVP (Platinol, 60 prine; ; Tiazole; Tice BGC; Tinzole (tizofurin); VP-16), pyrazine diazohydroxide; pyrazoloacridine, pyrido tirapazamine; Tiratricol; tizofurine; TLC D-99 (liposome glutethimide (Rogletimide); Quadramet; R-837; Radinyl encapsulated doxorubicin); TMCA, T-MOP (6-thioguanine, (etanidazole); Ralox, recombinant interleukin-2, Recombi methotrexate, Oncovin, prednisone); TMP (trimethoprim); nate; Renova (tretinoin emollient cream); Retin-A, retinoic TMQ (trimetrexate); TMTX (trimetrexate); TMX acid; R-Frone (interferon beta, Serono); Rheumatrex 65 (tamoxifen); TNF (tumor necrosis factor); TNP 470; TOAP (methotrexate sodium); RIDD (recombinant interleukin-2, (thioguanine, Oncovin, cytosine arabinoside, prednisone); dacarbazine, DDP); rIFN-A; rIFN-gamma; RIGS/ACT Tomosar (menogaril); Tomudex (ICI D1694); US 6,319,682 B1 35 36 (hy camp tamine); tore mife ne; TPCH (thioguanine, VIC (VP-16, ifosfamide, carboplatin); VIE (Vincristine, procarbazine, CCNU, hydroxyurea); TPDC-FUHU; ifosfamide, etoposide); vinblastine, Vincaleukoblastine; Vin TPDCV (thioguanine, procarbazine, DBC, CCNU, casar, Vincristine, Vindesline, Vinepidine, Vinglycinate; Vin Vincristine); TRA (trans-retinoic acid or tretinoin); trans leurosine; vinorelbine (Navelbine); Vinrosidine; Vinzolidine; retinoic acid (TRA); TRAP (thioguanine, rubidomycin, ara VIP (VePesid, ifosfaminde, Platinol); VIP-B (VP-16, C, prednisone); trestolone; tretinoin (all trans-retinoic acid); ifosfamide, Platinol, bleomycin); VLB (vinblastine); VLP triacana (tiratricol); tricinbine phosphate, triethylene (Vincristine, L-asparaginase, prednisone); VM-26 melamine; trifluoperazine, triloStane; trimetrexate, triptore (teniposide); VM-26PP (teniposide, procarbazine, lin; TSPA (thiotepa); TIPA (thiotepa); tubulozole; tumor prednisone); VMAD (Vincristine, methotrexate, necrosis factor (TNF); UFT (Uracil--Ftorafur); uracil mus 1O Adriamycin, actinomycin D); VMC (VP-16, methotrexate, tard; , uredepa; VA (Vincristine, Adriamycin); citrovorum factor); VMCP (Vincristine, melphalan, VAAP (Vincristine, asparaginase, Adriamycin, prednisone); cyclophosphamide, prednisone); VMP (VePesid, VAB 1 (vinblastine, actinomycin D, bleomycin); VAB 2 mitoxantrone, prednimustine); VOCA (VP-16, Oncovin, (vinblastine, actinomycin D, bleomycin, cisplatin); VAB 3 cyclophosphamide, Adriamycin); VOCAP (VP-16-213, (vinblastine, actinomycin D, bleomycin, cisplatin, 15 Oncovin, cyclophosphamide, Adriamycin, Platinol); Voro chlorambucil, cyclophosphamide); VAB 4 (vinblastine, acti zole; VP (vincristine, prednisone); VP-16; VP-16-213; nomycin D, bleomycin, cisplatin, cyclophosphamide); VAB VP-16+DDP (etoposide, cisplatin); VP-16-P (VP-16, 5 (vinblastine, actinomycin D, bleomycin, cisplatin, Platinol); VP-A (Vincristine, prednisone, asparaginase); cyclophosphamide); VAB 6 (cyclophosphamide, VPAM (verapamil); VPB (vinblastine, Platinol, bleomycin); dactinomycin, vinblastine, bleomycin, cisplatin); VABCD VPBCPr(vincristine, prednisone, vinblastine, chlorambucil, (vinblastine, Adriamycin, bleomycin, CCNU, DTIC); procarbazine); VPCA (Vincristine, prednisone, VAB-V (vinblastine, actinomycin D, bleomycin, cyclophosphamide, ara-C); VPCMF (vincristine, cyclophosphamide, cisplatin); VABCD (vinblastine, prednisone, cyclophosphamide, methotre Xate, Adriamycin, bleomycin, CCNU, DTIC); VAC (Vincristine, 5-fluorouracil); VP--L-asparaginase (Vincristine, actinomycin D, cyclophosphamide); VAC (Vincristine, 25 prednisone, L-asparaginase); VPP (VePesid, Platinol); Adriamycin, cyclophosphamide); VACA (Vincristine, acti VPVCP (Vincristine, prednisone, vinblastine, chlorambucil, nomycin D, cyclophosphamide, Adriamycin); VACAD procarbazine); Vulmon (teniposide); Wayne State protocol (Vincristine, Adriamycin, cyclophosphamide, actinomycin (cisplatin, 5-fluorouracil); Wehgen (estrone); Wellcovorin D, dacarbazine); VACP (Ve Pesid, Adriamycin, (leucovorin); Xomazyme-791 (anti-TAP-72 immunotoxin); cyclophosphamide, Platinol); VAD (vincristine, Xomazyme-H65 (CD5-T-lymphocyte immunotoxin); Adriamycin, dexamethasone); VAD/V (Vincristine, Zadaxin (thymosin alpha 1); Zanosar (streptozocin); Zanot Adriamycin, dexamethasone, Verapamil); VAFAC erone; ZD 0490; ZD 1694; ZDV (zidovudine); ZDX (Vincristine, amethopterin, 5-fluorouracil, Adriamycin (goserelin); Zeneca 182,780; zidovudine; Zinostatin; Zofran cyclophosphamide); VAI (Vincristine, actinomycin D, (ondansetron HCl); Zoladex (goserelin acetate), and ifosfamide); Valergen, VAM (VP-26-213, Adriamycin, 35 implant; Zolimomab aritoX (anti-T lymphocyte monoclonal methotrexate); VAMP (Vincristine, Adriamycin, antibody); ; Zorubicin HCl; Zyloprim. The present methotrexate, prednisone); VAMP (Vincristine, invention also contemplates high throughput Screening of amethopterin, 6-mercaptopurine, prednisone); VAP agents for treating psychiatric and neurological conditions (Vincristine, Adriamycin, procarbazine); VAP (Vincristine, and disorders. The following agents, and modifications of asparaginase, prednisone); VAP-II (vinblastine, actinomycin 40 the following agents, including including analogs, D, Platinol); vapreotide; VAT (vinblastine, Adriamycin, derivatives, fragments, active moieties, and the like, may be thiotepa); VAT (Vincristine, ara-A, 6-thioguanine); VATD Screened using methods and Systems of the present inven (Vincristine, ara-C, 6-thioguanine, daunorubicin); VATH tion: Adapin (Doxepin); Alprazolam; Amantadine, Amitrip (vinblastine, Adriamycin, thiotepa, Halotestin); VAV (VP tyline, Amoxapine; Anafranil (Clomipramine); Antabuse 26-213, Adriamycin, Vincristine); VB (vinblastine, 45 (Disulfiram); Artane (Trihexyphenidyl); Asendin bleomycin); VBA (vincristine, BCNU, Adriamycin); VBAP (Amoxapine); Ativan (Lorazepam), Aventyl (Nortriptyline); (Vincristine, BCNU, Adriamycin, prednisone); VBC Benadryl (Diphenhydramine); Benztropine; Bupropion; (VePesid, BCNU, cyclophosphamide); VBC (vinblastine, Buspar (Buspirone); Buspirone, Calan (Verapamil); Cal bleomycin, cisplatin); VBD (vinblastine, bleomycin, DDP); cium Carbimide; Carbamazepine; Carbolith (Lithium); Cel VBL (vinblastine); VBM (vincristine, bleomycin, DDP); 50 exa (Citalopram); Chlordiazepoxide; Chlorpromazine; VBMCP (v in cristine, BCNU, me 1 phalan, Cibalith-S (Lithium); Citalopram; Clomipramine; Clon cyclophosphamide, prednisone); VBMF (vincristine, azepam, Clozapine; Clo Zaril (Clozapine); Cogentin bleomycin, methotrexate, 5-fluorouracil); VBP (vinblastine, (Benztropine); Cylert (Pemoline); Dalmane (Flurazepam); bleomycin, Platinol); VC (VP-16, carboplatin); VCAP Depakene (Valproate); Desipramine, DeSyrel (Trazodone); (Vincristine, cyclophosphamide, Adriamycin, prednisone); 55 Dexedrine (Dextroamphetamine); Dextroamphetamine; VCAP-I (VP-16, cyclophosphamide, Adriamycin, Platinol); Diazapam; Dilantin (Phenytoin); Divalproex; Diphenhy VCF (vincristine, cyclophosphamide, 5-fluorouracil); dramine; Disulfiram; Doxepin; Duralith (Lithium); Edronax VCMP (Vincristine, cyclophosphamide, melphalan, (Reboxetine); Effexor (Venlafaxine); Elavil (Amitriptyline); prednisone); VCP (Vincristine, cyclophosphamide, Endep (Amitriptyline); Epitol (Carbmazepine); Epival prednisone); VCP-I (VP-16, cyclophosphamide, Platinol); 60 (Divalproex); Eskalith (Lithium); Ethosuximide; Etrafon VCR (vincristine); VDP (vincristine, daunorubicin, (Perphenazine); Fluanxol (Flupenthixol); Fluoxetine; Flu prednisone); VDS (Vindesine); VeeM-26 (teniposide); VelP penthixol; Fluphenazine, Flurazepam, Fluvoxamine, Hal (Velban, ifosfamide, Platinol); Velban (vinblastine sulfate); cion (Triazolam); Haldol (Haloperidol); Haloperidol; Imi Velsar (vinblastine sulfate); VEMP (Vincristine, Endoxan, pramine, Imovane (Zopiclone); Inderal (Propranolol); 6-mercaptopurine, prednisone); VePesid (etoposide or 65 Isoptin (Verapamil); Janimine (Imipramine); Klonopin VP-16); Vercyte (pipobroman); verteprofin; Vesanoid (all (Clonazepam), Lamotrigine; Lamictal (Lamotrigine); Lar trans-retinoic acid); VIC (vinblastine, ifosfamide, CCNU); gactil (Chlorpromazine); Libritabs (Chlordiazepoxide); Lib US 6,319,682 B1 37 38 rium (Chlordiazepoxide); Lithane (Lithium); Lithium; Nifedipine; Nitroglycerin; Norfloxacin; Nortriptyline; Nys Lithizine (Lithium); Lithobid (Lithium); Lithonate tatin; Ofloxacin; Omeprazole; Ondansetron; Oxybutynin; (Lithium); Lithotabs (Lithium); Lorazepam, Loxapac Oxycodone; Pediazole; Penicillin G, Pentamadine; Per (Loxapine); Loxapine, Loxitane (Loxapine); Ludiomil phenazine; Phenobarbital; Phenytoin; Piperacillin; Pred (Map rotiline); Luvox (Fluvoxamine); Manerix nisolone; Prednisone; Procainamide; Prochlorperazine; (Moclobemide); Maprotiline; Mellaril (Thioridazine); Promethazine; Propranolol; Pseudoephedrine; Pyrantel Mesoridazine; Methylphenidate; Moclobemide; Modecate pamoate; Ranitidine, Rifampin; Rimantadine, Salsalate; (Fluphenazine); Mysoline (Primidone); Nardil (Phenelzine); Sertraline, Simvastatin; Spironolactone, Succinylcholine; Navane; Nefazodone; Norpramine (Desipramine); Nortrip Sucralfate; Sumatriptan; Terazosin; Terbutaline; Tetracy tyline, Nozinan, Olanzapine; Orap; Oxazepam, Pamelor cline; Thiabendazole; Thiamine; Thioridazine; Ticarcillin; (Nortriptyline); Parnate (Tranylcypromine); Paroxetine; Ticlopidine; Tobramycin; Trazodone; Triazolam; Paxil (Paroxetine); Pemoline; Permitil (Fluphenazine); Per Trimethoprim/sulfamethoxazole; Valproic acid; Vecuro phenazine; Pertofrane (Desipramine); Phenelzine; Piportil nium; Venlafaxine; Verapamil; Warfarin; Zidovudine. (Pipotiazine); Pipotiazine; Primidone; Prolixin In one specific embodiment, a combination treatment for (Fluphenazine); Propranolol; Protriptyline; Prozac 15 Seizures and Seizure-related disorders, Such as epilepsy, may (Fluoxetine); Quetiapine; Reboxetine (Edronax); Restoril comprise an ion-dependent cotransporter antagonist, prefer (Temazepam); Rhotrimine (Trimipramine); Risperidal ably a cation-chloride cotransporter antagonist, adminis (Risperidone); Risperidone; Ritalin (Methylphenidate); tered in combination with one or more anti-convulsants or Rivotril (Clonazepam); Sabril (Vigabatrin); Serax anti-epileptic drugs using a delivery System that delivers the (Oxazepam); Serentil (Mesoridazine); Serocquel treatment composition(s) to the central nervous System. (Quetiapine); Sertraline; SerZone (Nefazodone); Sinequan Often the dose of the Second agent may be less than the (Doxepin); Stelazine (Trifluoperazine); Sulpiride; Surmontil Standard dosage as a consequence of the neurophysiological (Trimipramine); Symmetrel (Amantadine); T-Quil activity of the ion-dependent cotransporter antagonist. Illus (Diazapam); Tegretol (Carbamazepine); Temazepam, Tem trative Second agents for treatment in combination with the posil (Calcium Carbimide); Thioridazine; Thiothixene; 25 Subject compositions comprising ion-dependent antagonists, Thorazine (Chlorpromazine); Tofranil (Imipramine); Traz include, for example, phenytoin, carbamazepine, odone; Triazolam, Trifluoperazine, Trihexyphenidyl, barbiturates, phenobarbital, pentobarbital, mephobarbital, Trilafon (Perphenazine); Trimipramine; Triptil trime thadione, mephenytoin, parame thadione, (Protriptyline); Valium (Diazapam); Valium Injection phen the nylate, phen a c e mide, met harbital, (Dia Zapam), Valproate; Valproic acid; Valrelease ben Zchlorpropanmide, phensu Ximide, primidone, (Valproate); Venlafaxine; Verapamil; Viv actil methSuximide, ethotoin, aminoglutethimide, diazepam, (Protriptyline); Vigabatrin (Sabril); Wellbutrin (Bupropion); clonazepam, cloraZepate, foSphenytoin, ethoSuximide, Xanax (Alprazolam); Zarontin (Ethosuximide); Zoloft Valporate, felbamate, gabapentin, lamotrigine, topiramate, (Sertraline); Zopiclone; Zyprexa (Olanzapine). In addition, Vigrabatrin, tiagabine, Zonisamide, clobazam, thiopental, the following agents, and modifications of the following 35 midazoplan, propofol, levetiracetam, Oxcarbazepine, agents, including, for example, analogs, derivatives, CCPene, GYK152466 and Sumatriptan. As can be readily fragments, active moieties, and the like, may be Screened appreciated, the above-noted compounds are only examples using methods and Systems of the present invention: of Suitable treatment combinations, and other compounds or Acetaminophen; Acyclovir, Albuterol (oral); Albuterol, Similar classes of compounds are also Suitable. The activity inhaled; Allopurinol; Alprazolam; Amantadine, Amikacin; 40 and efficacy of Such compounds, and various combinations Amitriptyline, Amlodipine, Amoxicilin; Amoxicillin/ and dosages of Such compounds and combinations may be clavulanate, Ampicillin; Aspirn, Atenolol, Azithromycin; assessed using the methods and Systems of the present Aztreonam, Beclomethasone, BenZonatate; Benztropine; invention. Bethanechol; Bumetanide, Captopril; Carbamazepine, Cefa In one exemplary embodiment, candidate agent combi clor, Cefadroxil, Cefamandole; Cefazolin; Cefixime; Cefo 45 nations for treating epilepsy are tested using Screening taxime; Cefoxitin; Ceftazidime; Ceftizoxime; Ceftriaxone; methods of the present invention. Agents having activity as Cephalexin; Chlorpromazine, Cimetidine, Ciprofloxacin; a chloride cotransporter agonist and/or antagonist may be Cisapride; Clarithromycin; Clindamycin; Clonidine, identified using methods and Systems of the present inven Codeine; Colchicine, Desipramine, Dexamethasone; Dex tion. Candidate compounds may be Screened for chloride tromethorphan; Diazepam, Dicloxacillin; Digoxin; Digoxin 50 cotransporter agonist and/or antagonist activity using Fab, Diltiazem; Diphenhydramine; Dipyridamole; Dival Screening methods of the present invention with various proex, Doxycycline, Droperidol, Enalapril, Enoxaparin; types of cells in culture Such as glial cells, neuronal cells, Epinephrine; Epinephrine in; Sesame oil, (Sus-Phrine); renal cells, and the like, or in Situ in animal models. Erythromycin; Estrogen, conjugated; Ethacrynic acid; Screening techniques to identify chloride cotransporter Ethosuximide; Famciclovir, Famotidine, Felbamate; 55 antagonist activity, for example, may involve altering the Flconazole, Flumazenil; Fluoxetine; Folic acid; Furosemide; ionic balance of the extracellular Space in the tissue culture Gabapentin; Gentamicin; Glipizide, Glucagon; Glyburide; Sample, or in Situ in an animal model, by producing a higher Griseofulvin; Haloperidol; Heparin; Hydrochlorothiazide; than “normal” anionic chloride concentration. The geometri Hydrocortisone; Hydroxy Zine, Ibuprofen; Imipramine; cal and/or optical properties of the cell or tissue Sample Indomethacin; ISOSorbide dinitrate; Ketorolac, Labetalol; 60 Subject to this altered ionic balance are determined, and Lactulose; LevothyroXine, Lidocaine, Lorazepam, Lovasta candidate agents are administered. Following administration tin, Magnesium oxide, Magnesium Sulfate; Mebendazole; of the candidate agents, the corresponding geometrical and/ Meclizine; Medroxyprogesterone; Mefenamic acid; Meperi or optical properties of the cell or tissue Sample are moni dine; Methicillin; Methylergonovine; Methylphenidate; tored to determine whether the ionic imbalance remains, or Methylprednisolone; Metoclopramide; Metolazone (Diulo; 65 whether the cells responded by altering the ionic balances in and Zaroxolyn only); Metoprolol; Metronidazole; Mida the extracellular and intracellular space. If the ionic imbal Zolam; Morphine sulfate; Nafcillin; Naloxone; Naproxen; ance remains, the candidate agent is likely a chloride US 6,319,682 B1 39 40 cotransporter antagonist. By Screening using various types 2 mM EGTA (four slices, three animals). In all treatments, of cells or tissues, candidate compounds having a high level perfusion with furosemide-containing medium was begun of glial cell chloride cotransporter antagonist activity and after a consistent level of bursting had been established. having a reduced level of neuronal cell and renal cell For imaging of intrinsic optical Signals, the tissue was chloride cotransporter antagonist activity may be identified. illuminated with a beam of white light (tungsten filament Similarly, effects on different types of cells and tissue light and lens system; Dedotec USA, Lodi, N.J.) directed Systems may be assessed. through the microscope condenser. The light was controlled Additionally, the efficacy of candidate compounds for and regulated (power Supply: Lambda Electronics, Melville, treating various conditions of the central and peripheral N.Y.) to minimize fluctuations and filtered (695 nm long nervous System may be assessed by Simulating or inducing pass) So that the slice was transilluminated with long wave a condition, Such as a Seizure, central nervous System edema, lengths (red). Image frames were acquired with a charge ethanol neurotoxicity, cortical Spreading depression, or the coupled device camera (Dage-MTI) at 30 Hz and were like, in a tissue Sample or in Situ in an animal model, digitized at 8 bits with a spatial resolution of 512 by 480 monitoring the geometrical and/or optical properties of the pixels by means of an Imaging Technology Series 151 cell or tissue Sample during Stimulation of the condition, 15 imaging System, gains and offsets of the camera-control box administering the candidate compound, then monitoring the and the analog-to-digital board were adjusted to optimize the geometrical and/or optical properties of the cell or tissue Sensitivity of the System aging hardware was controlled by Sample following administration of the candidate a 486-PC-compatible computer running software written by compound, and comparing the geometrical and/or optical D. Hochman and developed with commercially available properties of the cell or tissue Sample to determine the effect software tools (Microsoft's C/C++ Compiler and Imaging of the candidate compound. Similarly, the efficacy of treat Technology's ITEX library). To increase signal-to-noise ment composition(s) in an animal or human Subject may be ratio, an averaged image was composed from 16 individual monitored in Situ using the optical methods and Systems of image-frames, integrated over 0.5 S and averaged together. the present invention. An experimental Series typically involved the continuous According to yet another embodiment, Screening methods 25 acquisition of a Series of averaged images over a Several of the present invention may be accomplished by performing minute time period; at least 10 of these averaged images different Steps at remote locations. Thus, in Vitro and in vivo were acquired as control images before Stimulation. Pseudo Screening data may be collected at one location, Such as in colored images were calculated by Subtracting the first a testing laboratory. The data relating to optical and/or control image from Subsequently acquired images and geometrical properties of cell Sample populations at various assigned a color lookup table to the pixel values. For these test Stages may be collected and transmitted to a central data images, usually a linear low-pass filter was used to remove processor for data processing and analysis, and Screening high-frequency noise and a linear-histogram Stretch was results may Subsequently be transmitted to the testing labo used to map the pixel values over the dynamic range of the ratory or to one or more additional Sites. Network and System. All operations on these images were linear So that communications technologies are readily available for trans 35 quantitative information was preserved. mitting the required data and implementation of remote FIGS. 2A-2C show the effect of the agent furosemide on activities, involving data processing and/or analysis, may Stimulation evoked afterdischarge activity in a hippocampal provide expedited Screening results and more economical tissue Slice comparing the field response, measurements at Screening. an extracellular electrode, and images highlighting changes The following Examples are provided for illustrative 40 in optical properties. purposes only and are not intended to limit the invention in FIG. 2A1 illustrates that two seconds of electrical stimu any way. lation at 60 Hz elicited afterdischarge activity. FIG. 2A2 shows a typical afterdischarge episode recorded by the EXAMPLE 1. extracellular electrode, with the horizontal arrow indicating Sprague-Dawley rats (male and female; 25 to 35 days old) 45 the baseline. FIG. 2A3 shows a map of the peak change in were prepared as described in Aghajanian, A. K. and optical transmission through the tissue evoked by Schaffer Rasmussen, K., Synapse 31:331, 1989; and Buckmaster, P. collateral Stimulation. The color bar indicates increasing S., Strowbridge, B. W., Schwartzdroin, P. A., J. Neuro magnitude of activity-evoked optical changes from the bot physiol. 70: 1281, 1993. In most hippocampal slice tom to the top of the bar. The region of maximum optical experments, Simultaneous extracellular field electrode 50 change (red, yellow) corresponds to the apical and basal recordings were obtained from CA1 and CA3 areas. For dendritic regions of CA1 on either Side of the Stimulating Stimulation-evoked afterdischarge (13 Slices, 8 animals), the electrode. FIG. 2B1-2BC illustrate responses to electrical concentration of Mg" in the bathing medium was reduced stimulation following 20 minutes of perfusion with medium to 0.9 mM. A bipolar tungsten stimulating electrode was containing 2.5 mM furosemide. Both the electrical afterdis placed on the Schaffer collaterals to evoke Synaptically 55 charge activity (shown in FIG. 2B2) and the stimulation driven field responses in CA1; stimuli consisted to 100 to evoked optical changes (shown in FIG. 2BC) were blocked. 300-us-duration pulses at an intensity of four times However, there was a hyperexcitable field response population-Spike threshold. After discharges were evoked by (multiple population spikes) to the test pulse, as illustrated a 2-S train of such stimuli delivered at 60 Hz. Spontaneous in FIG. 2B1. FIGS. 2C1-2C3 illustrate that restoration of the interictal-like bursts were observed in slices treated with the 60 initial response pattern was seen following 45 minutes of following modifications or additions to the bathing medium: perfusion with normal bathing medium. 10 mM K" (6 slices; 4 animals; average, 81 bursts/min), 200 FIG. 3A illustrates an enlarged grey-Scale image of an to 300 uM 4-AP (4 slices; 2 animals; average, 33 bursts/ acute rat hippocampal tissue Slice, observed using a CCD min), 50 to 100 ug M bicuculine (4 slices; 3 animals; camera attached to a Zeiss upright microscope. FIGS. average, 14 bursts/min), 0 mM Mg" (1 hour of perfusion) 65 3B-3E illustrate enlarged, contrast-enhanced images 3 Slices, 2 animals; average, 20 bursts/min, (3 hours of acquired as described above. FIG.3B illustrates an enlarged, perfusion) 2 slices, 2 animals), 0 mM Ca"/6 mM KCI and contrast-enhanced image acquired as described above dur US 6,319,682 B1 41 42 ing the peak optical change induced by electrical Stimulation trials. The average percentage changes over the Stimulation, with an enlarged bar, the arrow on the bar four areas (indicated by the four Square regions marked in indicating increasing magnitude of activity-evoked optical FIG. 4A) were plotted graphically in FIGS. 4B, 4C, and 4D changes. The box indicates the field of view shown magni for comparison and analysis of the dynamic changes occur fied in FIGS. 3C, 3D and 3E. FIG. 3C illustrates the peak ring in these four Spatial areas. optical change during electrical Stimulation when no epilep The optical changes between the Stimulating electrodes tic activity was induced. FIG. 3D illustrates the peak optical (Site #1, FIG. 4A) and near the recording electrode (Site #3) change during electrical Stimulation that resulted in epilep showed a graded response to the intensity and duration of tiform activity. A larger area of increased magnitude of each afterdischarge episode (FIG. 4B). The spatial extent of changes in optical properties is observed during epileptiform 1O the epileptiform activity was demonstrated by comparing a activity. FIG. 3E illustrates the peak optical change during baseline image collected before Stimulation to those electrical Stimulation following treatment with furosemide, obtained immediately after stimulation. The intensity and which blocks the epileptiform activity and the intrinsic Spread of the optical changes were much less following optical Signal. Stimulation #2 (shortest, least intense afterdischarge episode) than after Stimulation #4 (longest, most intense EXAMPLE 2 15 afterdischarge episode). This example illustrates optical changes indicative of When the optical changes were below baseline, the Sur neuronal activity in a human Subject by direct cortical face EEG recordings did not identify epileptiform activity electrical Stimulation. Surface electrical recordings (Surface (n=3 patients). At Site #3, the optical changes after stimu EEG, ECOG) were correlated with optical changes. Intrinsic lation were below baseline. However, during the fourth optical changes were evoked in an awake patient during Stimulation, the epileptiform activity Spread into the area of stimulating-electrode “calibration.” Four stimulation trials Site #3 and the optical signal did not go below baseline until were Sequentially applied to the cortical Surface, each Stimu later. This negative optical Signal likely represents inhibited lation evoking an epileptiform afterdischarge episode. A neuronal populations (an epileptic inibitory Surround), Stimulation trial consisted of: (1) monitoring resting cortical decreased oxygen delivery, or blood Volume shunted to activity by observing the output of the recording electrodes 25 activated regions. for a brief period of time; (2) applying an electric current via FIG. 4B shows plots of the percent optical change per the Stimulation-electrodes to the cortical Surface at a par Second in the spatial regions of Boxes 1 and 3 (as labeled in ticular current for several Seconds, and (3) monitoring the FIG. 4A). For both regions, the peak change is during the output of the recording electrodes for a period of time after fourth stimulation trial (at 8 mA), in which the greatest Stimulation has ceased. amount of Stimulating current had induced the most pro The cortex was evenly illuminated by a fiber optic emir longed epileptiform afterdischarge activity. The changes passing through a beam splitter, controlled by a D.C. regu within Box3 were greater and more prolonged than those of lated power Supply (Lambda, Inc.) and passed through a 695 Box 1. Box 3 was overlying the area of the epileptic focus. nm longpass filter. Images were acquired with a CCD 35 FIG. 4C ShowS plots of the percent optical change per camera (COHU 6500) fitted to the operating microscope Second in the spatial regions of Boxes 1 and 4 (as labeled in with a specially modified cineadaptor. The cortex was Sta FIG. 4A). Box 1 overlays an area of cortical tissue between bilized with a glass footplate. Images were acquired at 30 HZ the two Stimulating electrodes, and Box 4 overlays a blood and digitized at 8 bits (512x480 pixels, using an Imaging vessel. The optical changes within box 4 are much larger and Technology Inc. Series 151 system, Woburn, Mass.). Geo 40 in the opposite direction of Box 1. Also, these changes are metric transformations were applied to images to compen graded with the magnitude of Stimulating current and after sate for small amounts of patient motion (Wohlberg, Digital discharge activity. The changes in Box 4 are most likely due Imaging Warping, IEEE Computer Society: Los Alamitos, to changes of the blood-flow rate within a blood vessel. Calif., 1988). Subtraction of images collected during the FIG. 4D ShowS plots of the percent optical change absorp Stimulated State (e.g., during cortical Surface Stimulation, 45 tion per Second in the Spatial regions of boxes 1 and 2 (as tongue movement or naming) from those collected during a labeled in FIG. 4A). Note that although these two areas are control State with Subsequent division by a control image nearby each other, their optical changes are in the opposite resulted in percentage difference maps. Raw data (i.e., no direction during the first three Stimulation trials using 6 mA digital enhancement) were used for determining the average current. The negative going changes within the region of optical change in Specified regions (average sized boxes was 50 Box 2 indicate that the methods and apparatus of the present 30x30 pixels or 150–250 um'). For pseudocolor images, a invention may be used to monitor exhibition of physiologi linear low pass filter removed high frequency noise and cal changes, as well as excitation. linear histogram transformations were applied. Noise was FIG. 5 shows percentage difference imageS representative defined as the Standard deviation of fluctuations in Sequen of various times during two of the Stimulation trials tially acquired control images as 0.003–0.009. 55 described above. The top three images (5A2, 5B2, and 5C2) A Series of images (each image consisting of an average are from Stimulation Trial 2, where 6 mA cortical stimula of 128 frames acquired at 30 Hz) were acquired during each tion evoked a brief period of afterdischarge. These are of the four stimulation trials. A current of 6 mA was used for compared to the bottom three images (5A4, 5B4, and 5C4), the first three stimulation trials, and 8 mA for the fourth. which are from Stimulation Trial 4, showing the optical After a sequence of 3-6 averaged control images were 60 changes evoked by cortical stimulation at 8 mA. FIGS. 5A2 acquired, a bipolar cortical Stimulation current was applied and 5A4 compare control images during rest. FIGS. 5B2 and (either 6 mA or 8 mA) until epileptiform after discharge 5B4 compare the peak optical changes occurring during the activity was evoked (as recorded by the Surface electrode). epileptiform afterdischarge activity. FIGS. 5C2 and 5C4 Images were continuously acquired throughout each of the compare the degree of recovery 20 Seconds after the peak four Stimulation trials. 65 optical changes were observed. The magnitude of optical The percentage change in absorption of light for each change is indicated by the grey-Scale changes. Each image pixel was calculated for each image acquired during the four maps an area of cortex approximately 4 cm by 4 cm. US 6,319,682 B1 43 44 FIGS. 6A-6H show eight percentage difference images pression of baseline noise in Surrounding areas indicated from Stimulation Trial 2, as described above. Each image is that, during the tongue Wiggling trials, language-motor areas integrated over a two Second interval. The focal area of showed a negative-going optical Signal. Image 8B2 is per greatest optical change is in the center of imageS3C, 3D, and centage difference control image of the cortex during one of 3E, indicating the region of greatest cortical activity. This the language naming trials. Image 8B3 is a percentage region is the epileptic focus. The magnitude of optical difference image of the peak optical change in the cortex change is indicated by the grey-Scale bar on the right Side of during the language naming task. Large positive-going Sig the Figure. The arrow beside this grey-Scale indicates the nals are present in Broca's area. Negative-going Signals are direction of increasing amplitude. Each image maps an area present in tongue and palate Sensory areas. of cortex approximately 4 cm by 4cm. FIG. 9 shows time course and magnitude plots of dynamic FIGS. 7A-7H illustrate a real-time sequence of dynamic optical changes in human cortex evoked in tongue and palate changes of Stimulation-evoked optical changes in human Sensory areas and in Broca's area (language). This figure cortex. FIG. 4, panels 4A through 4H, Show eight consecu shows the plots of the percentage change in the optical tive percentage difference images. Each image is an average absorption of the tissue within the boxed regions shown in of 8 frames (

PATENT NO. : 6,319,682 B1 Page 1 of 2 DATED : November 20, 2001 INVENTOR(S) : Daryl W. Hochman

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 6 Line 15, replace “contrast-enchanced with -- pseudo-colored -- Column 9 Line 53, replace" 99 Case 3: AA=-45,(x,x)+(y-5x)(x-x): with -- Case 3: AA = -(%5, (x,-1-X) + (y –5, xi) (xi. -x) . Column 21 Line 12, replace “actinomcin-D” with -- actinomycin-D -- Line 19, replace "prdnisone)” with -- prednisone) --

Column 39 Line58, replace “After discharges” with -- Afterdischarges --

Column 40 Line 17, replace "System aging hardware' with -- System. Imaging hardware -- Line 67, replace “contrast-enhanced” with -- pseudo-colored -- Column 41 Line 2, replace “with an enlarged bar, the arrow on the bar” with -- with an enlarged color bar, the arrow on the color bar -- Column 46 Lines 21-22, replace “inmumodulatory agents' with -- immunomodulatory agents -- Lines 22-23, replace "apostolic agents' With -- apoptotic agents --

Column 47 Line 29, replace “inmumodulatory agents' with -- immunomodulatory agents -- Line 30, replace "apostolic agents' with -- apoptotic agents -- UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,319,682 B1 Page 2 of 2 DATED : November 20, 2001 INVENTOR(S) : Daryl W. Hochman

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 48 Line 41, replace "of a transgenica or plant' with -- of a transgenic animal or plant --

Signed and Sealed this Twenty-seventh Day of August, 2002

Attest.

JAMES E ROGAN Attesting Officer Director of the United States Patent and Trademark Office