Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material

Definitions

• the present invention relates, in general, to thermography and, in particular, to a method of using infrared thermography to monitor physiological and molecular events that elicit a thermogenic response in animals (including humans), plants, tissues, cells and cell-free systems.
• the present method can be used for screening, identifying, and ranking drug candidates for multiple diseases, disorders and conditions.
• thermogenesis Thermodynamics is a science concerned with relations between work and heat. Virtually every chemical reaction or physiological process in animals or cells occurs with the absorption or generation of heat and thus, any heat absorbed or generated by a system is proportional to the amount of work done. Consequently, measurement of heat output (i.e., thermogenesis) can be used to estimate the energy used in or produced by chemical reactions and physiological processes.
• GDP Guanosine 5'-diphosphate
• fluorescent dyes e.g., JC-1 or rhodamine derivatives
• Infrared thermometers have been developed that can measure the magnitude of infrared energy emitted from a specific body site (e.g., the ear canal). These instruments, however, cannot be used to measure heat production of isolated cells, tissues, or chemical reactions and cannot provide real time measurements of heat output by multiple samples over extended periods of time. Moreover, these devices do not provide images over large surface areas. Infrared interactance instruments have also been developed. Unlike infrared thermometers, these instruments contain diodes that emit near radiation at wavelengths of ⁇ 1000 nm. Since these instruments measure the absorption of near infrared radiation, they do not provide an accurate measure of thermogenesis.
• the present invention provides a rapid non-invasive method of measuring real-time thermogenesis in animals, plants, tissues and isolated cells, including cells in culture.
• This invention extends to molecular interactions, such as receptor-ligand binding, enzyme catalysis, and other chemical reactions that alter heat output.
• the present method which is based on the use of infrared thermography, can be used to screen and identify drug candidates for treating various diseases, disorders and conditions. .
• the present invention relates generally to a method of monitoring physiological changes and molecular interactions using infrared thermography.
• Infrared thermography provides a non-invasive approach to analyze the effects of any of a variety of agents on heat production in animals, plants, cells in culture, and chemical reactions in cell-free systems.
• the invention makes it possible to screen compounds for their ability to alter heat dissipation, and to identify compounds that have application in the treatment of various diseases, disorder and conditions. Objects and advantages of the present invention will be clear from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS
• Figure 1 Schematic of an apparatus suitable for use in imaging infrared thermogenesis in cells in culture.
• FIG. 1 Schematic of an infrared thermography device suitable for use in imaging thermogenesis in a living animal.
• FIG. 3A Infrared thermography data of yeast (Fig. 3A;
• FIGs 4A-4C Presentation of thermographic analysis of yeast cells expressing uncoupling protein 2 (UCP2) (Figs. 4A and 4B) and molecular analysis of UCP2 expression in the yeast cells (Fig. 4C).
• UCP2 uncoupling protein 2
• thermographic analysis of thermogenesis in Chinese hamster ovary cells (CHO) overexpressing the ⁇ 3 -AR receptor in the presence of forskolin (- ⁇ -) or isoproterenol (-•-) (blank -A-).
• FIG. 7 Infrared thermographic image of differentiating adipocytes representing a dose response curve for several peroxisome proliferator activated receptor ⁇ (PPAR ⁇ ) agonists in the presence of insulin and 9-c/s retinoic acid.
• PPAR ⁇ peroxisome proliferator activated receptor ⁇
• a PPAR ⁇ agonist GW1929
• MED minimal effective dose
• Figure 9 Presentation showing rotenone sensitivity of ⁇ -AR agonist (50nM)-induced thermogenesis in adipocytes.
• VEGF vascular endothelial growth factor
• FIGs 13A-13D Infrared thermographic analysis of a metered dose inhaler (MDI) device (Fig. 13A; actuations 0, 1 and 5) during and after 5 consecutive actuations (Fig. 13B). Presentation of infrared thermographic analysis of nude mice treated with the inhalant, Albuterol (Fig. 13 C), and subsequent quantitation of the thoracic areas showing the kinetics of Albuterol activity (Fig 13D).
• MDI metered dose inhaler
• FIGs 18A and 18B Infrared thermographic analysis of diet- induced thermogenesis in humans.
• Fig. 18A shows dosal temperature versus time;
• Figure 22 Infrared thermographic analysis of the effect of anti- VEGF antibody on tumor temperature.
• FIGS 23A and 23B Infrared thermographic analysis of the effect of etoposide treatment (6 mg/kg) in utero on hair loss in newborn mouse pups.
• Fig. 23A shows the thermographic image obtained at day 0, day 3 and day 5.
• Figure 24 Infrared thermographic analysis of Pinacidil-induced changes in thermogenesis in rat genitalia (2 hours post PO dosing).
• the present invention is directed to a method of using infrared thermography as a tool to monitor temperature changes that occur during molecular interactions, including those that occur in isolated cells, tissues, plants, animals (e.g., man).
• Infrared thermography can be used to analyze the effects of various agents on heat production in a variety of cell, tissue, plant, and animal types, during enzyme catalysis, and, more generally, during ligand interaction with a binding partner.
• infrared thermography can be used to identify agents suitable for treating various diseases, disorders, and conditions, including those involving altered thermogenic responses.
• infrared radiation refers to electromagnetic radiation having a wavelength of between about 2.5 and about 50 microns or, expressed differently, that having a frequency of between about 200 and about 4000 inverse centimeters (cm “1 or “wave numbers”).
• IR radiation infrared
• the frequencies of electromagnetic radiation generally characterized as infrared are emitted or absorbed by vibrating molecules, and such vibrations generally correspond to the thermal state of a material in relation to its surroundings. All solid bodies whose temperatures are above absolute zero radiate some infrared energy, and for temperatures up to about 3500°K (3227° Celsius, 5840° Fahrenheit), such thermal radiation falls predominantly within the infrared portion of the electromagnetic spectrum.
• the monito ⁇ ng of radiation in the range of 3-100 microns is preferred, 3-15 microns being more preferred and 3-12 microns being most preferred (e.g., 6-12 microns).
• wavelengths below 2.5 cm “1 are considered as the "near IR” portion of the electromagnetic spectrum, and represent vibrational
• an infrared imaging system advantageously, a high resolution infrared imaging system, is used to monitor real time heat output, for example, from cells or tissues in culture or from laboratory animals, with images provided by a central processing unit for data analysis (see Figs. 1 and Fig. 2 and Examples that follow).
• a suitable system is that produced by FLIRInfrared Systems (the AGEMA 900 or QWIP SC3000).
• the temperature can be measured using a non-contact infrared thermometer such as that produced by Linear Laboratories (the C-1600MP). Neither the AGEMA 900 nor the C-1600MP has heretofore typically been used in to practice a method of the present invention.
• these apparatuses can be adapted by techniques known to those skilled in the art to measure changes in temperature in the range of, for example, 5.0°C, or higher, to 0.000001 °C or lower, preferably, 1.0°C to 0.00001 °C or 0.5°C to 0.0001 °C, more preferably, 0.3°C to 0.0005°C or 0.25°C to 0.001 °C, most preferably, 0.2°C to 0.002°C (see Fig. 1 and Fig. 2 and Examples that follow).
• the present invention relates to a method of monitoring the effect of an agent on thermogenesis in isolated cells, tissues, or in animals (including primates (e.g., humans)).
• the method comprises: i) measuring the heat produced by the cells, tissues, or a given surface area of animals before exposure to the agent using infrared thermography, ii) exposing the cells, tissues, or animal to the agent (e.g., by adding the agent to culture medium in which the cells or tissues are maintained/grown or by treating the animal with the agent using standard delivery techniques), iii) measuring the heat produced by the cells, tissues, or animals during and/or after treatment with the agent using infrared thermography, and iv) comparing the measurements obtained in steps (i) and (iii), wherein an agent that results in a lowering of the temperature of the cells, tissues or animals is an inhibitor of thermogenesis and an agent that results in an elevation of the temperature is a stimulator of thermogenesis.
• Cells that can be monitored in accordance with the invention include isolated naturally occurring cells (including primary cultures and established cell lines) and engineered cells (e.g., isolated engineered cells).
• the cells can be in suspension or attached to a solid support either as a monolayer or in multilayers.
• suitable supports include plastic or glass plates, dishes or slides, membranes and filters, flasks, tubes, beads and other related receptacles.
• plastic multiwell plates are used, 96-well and 384-well microtiter plates being preferred. While preferred cell titers range between 100 to 100,000 cells/cm 2 for adherent cells and 100 to 1 ,000 cells/ ⁇ l in the case of suspension cultures, potentially any cell number/concentration can be used.
• Isolated naturally occurring cells that can be monitored in accordance with the present method include eucaryotic cells, preferably mammalian cells.
• Primary cultures and established cell lines and hybridomas can be used. Specific examples include cells or tissues derived " from fat (e.g., adipocytes and precursors thereof), muscle (e.g., myotubes, myoblasts, myocytes), liver (e.g., hepatocytes, Kupffer cells), the digestive system (e.g., intestinal epithelial, salivary glands), pancreas (e.g., ⁇ and ⁇ -cells), bone marrow (e.g., osteoblasts, osteoclasts, and precursors thereof), blood (e.g., lymphocytes, fibroblasts, reticulocytes, hematopoietic progenitors), skin (e.g., keratinocytes, melanocytes), amniotic fluid or placenta (e.g., keratinocytes,
• the present method is applicable to cells derived from plants, fungi, protozoans, and the monera kingdom (e.g., bacteria).
• the cells can be cultured using established culture techniques and culture conditions can be optimized to ensure viability, growth and/or differentiation, as appropriate.
• Engineered cells that can be monitored in accordance with the present method include cells engineered to produce or overproduce proteins involved directly or indirectly in temperature regulation, energy balance and fuel utilization, growth and differentiation and other aspects of physiology or metabolism that alter heat generated by cells.
• Such cells can be engineered prokaryotic cells (kingdom monera: e.g., E. coli), engineered higher or lower eucaryotic cells, or cells present in or isolated from transgenic animals.
• Examples of higher eucaryotic cells include cell-lines available from the American Type Culture Collection (e.g., CV-1 , COS-2, C3H10T1/2, HeLa, and SF9).
• lower eucaryotic cells include fungi (e.g., yeast) and protozoans (e.g., slime molds and ciliates).
• the cells or transgenic animals can be engineered to express any of a variety of proteins, including but not limited to nuclear receptors and transcription factors (e.g., retinoid receptors, PPARs, CCAAT-Enhancer-Binding Proteins (CEBPs), polymerases), cell surface receptors (e.g., transmembrane and non-transmembrane receptors, G protein-coupled receptors, kinase- coupled receptors), membrane transporters and channels (e.g., uncoupling proteins, sugar transporters, ion channels), signal transduction proteins, (e.g., phosphodiesterases, cyclases, kinases, phosphatases), and viruses (e.g., AIDS, herpes, hepatitis, adeno).
• nuclear receptors and transcription factors e.g
• Engineered cells can be produced by introducing a construct comprising a sequence encoding the protein to be expressed and an operably linked promoter into a selected host .
• Appropriate vectors and promoters can be selected based on the desired host and introduction of the construct into the host can be effected using any of a variety of standard transfection/transformation protocols (see Molecular Biology, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Press, 1989).
• Cells thus produced can be cultured using established culture techniques and culture conditions can be optimized to ensure expression of the introduced protein-coding sequence.
• the present method can be used to identify, characterize, rank, and select agents (e.g., drugs or drug candidates) suitable for use in treating various diseases, disorders or conditions based on potency, selectivity, efficacy, pharmacokinetics and pharmacodynamics of the agent in various cell-free, cell, tissue, plant, animal, and human-based thermogenesis assays.
• agents e.g., drugs or drug candidates
• a test agent can be screened using infrared thermography for its potential as a catabolic or anabolic drug.
• Cultured cells e.g., primary cells, such as adipocytes or yeast, or cell- lines, such as C3H10T1/2 mesenchymal stem cells, osteoblasts, or adipocytes
• plants, animals, or humans including patients in clinical studies during pharmaceutical development
• infrared thermography to measure changes in heat signature.
• Agents that enhance thermogenesis (cellular heat production) are potentially useful as catabolic drugs and agents that suppress thermogenesis are potentially useful as anabolic drugs.
• thermogenesis can reflect changes in growth and differentiation.
• the present method can be used to identify, characterize, rank, and select agents (e.g., drugs or drug candidates) suitable for use in treating or preventing diseases, disorder or conditions associated with changes in metabolism, toxicity, cellular growth, organ development, and/or differentiation.
• agents e.g., drugs or drug candidates
• pathophysiologies potentially amenable to treatment with anabolic agents identified with infrared thermography include anorexia, alopecia, auto-immunity, cachexia, cancer, catabolism associated with aging, diabetes, graft rejection, growth retardation, osteoporosis, pyrexia, bacterial and viral infections.
• diseases, disorders or conditions potentially amenable to treatment with catabolic agents identified with infrared thermography include diseases, disorders or conditions associated with obesity (e.g., hypertension, dyslipidemias, and cardiovascular diseases) and diseases, disorders or conditions associated with accelerated growth (e.g., cancer, gigantism, certain viral infections).
• the pathophysiologies amenable to treatment using agents identified with infrared thermography are not limited to those commonly associated with changes in anabolism or catabolism (e.g., metabolic diseases).
• the approach is also applicable to other diseases, disorders and conditions including male erectile dysfunction (MED), inflammation, hypertension, gastrointestinal diseases, behavorial disorders (CNS diseases), and diseases associated with changes in blood flow.
• MED male erectile dysfunction
• CNS diseases behavorial disorders
• diseases associated with changes in blood flow There are no restrictions on the pathophysiologies that can be analyzed in accordance with the present invention in pharmaceutical research and development (e.g.., analysis of drug potency, efficacy, toxicity
• the binding of a ligand (proteinaceous or nonproteinaceous (e.g., a nucleic acid)) to a binding partner (proteinaceous or nonproteinaceous (e.g., a nucleic acid)), where binding elicits a thermogenic response can be monitored using infrared thermography.
• the ligand and/or binding partner can be in a cell or in a cell-free environment (e.g., a solution).
• the ligand and/or binding-partner can be a synthesized chemical entity that does not normally exist in nature, or the ligand and/or binding-partner can be a naturally occurring entity such as a naturally occurring protein, nucleic acid, polysaccharide, Iipid, hormone, or other naturally occurring substance or cell.
• a test agent e.g., potential ligand
• the effect of a test agent on heat generated by its binding partner can be measured using infrared thermography.
• One suitable method comprises: i) measuring the heat produced by the binding- partner, ii) adding test agent to the binding-partner, iii) measuring heat produced after mixing the potential ligand (test agent) and binding-partner, and iv) comparing the measurements in (i) and (iii), wherein an agent that alters heat generation is a ligand for the binding partner.
• test agents can be screened for their ability to alter the thermogenic response resulting from the binding of the ligand to its binding-partner.
• agents can be allosteric regulators, agonists, or antagonists of the ligand and/or binding partner.
• Such a screen can comprise: i) measuring the heat produced upon addition of the first member of the binding pair (ligand or binding-partner) to the second member of the binding-pair using infrared thermography, and ii) measuring the heat produced upon addition of the first member of the binding pair, the second member of the binding-pair and test agent, and iii) comparing the measurement in (i) with that in (ii), wherein an agent that alters the heat generation observed upon addition of the ligand to its binding partner is a modulator of that interaction, for example, by binding to either or both members of the binding pair.
• agents can be screened for their ability to modulate the rate of catalysis of a particular enzyme.
• the method can comprise measuring the heat produced upon addition of an enzyme to its substrate using infrared thermography and measuring the heat produced upon addition of a test agent, the enzyme, and its substrate, and comparing the results.
• An agent that alters heat production can be an enzyme inhibitor or activator.
• Controls that can be run in accordance with such a method include measuring the heat produced upon addition of the enzyme to the test compound (in the absence of substrate) and upon addition of the substrate to the test compound (in the absence of the enzyme). Such controls permit determination of the effects on heat production from the respective additions.
• test agents can be screened for their ability to behave as substrates.
• the present invention relates to agents identified using the above-described assays.
• the agents identified in accordance with the above assays can be formulated as pharmaceutical compositions.
• Such compositions comprise the agent and a pharmaceutically acceptable diluent or carrier.
• the agent can be present in dosage unit form (e.g., as a tablet or capsule) or as a solution, preferably sterile, particularly when it is to be administered by injection.
• the dose and dosage regimen will vary, for example, with the patient, the agent and the desired effect. Optimum doses and regimens can be determined readily by one skilled in the art.
• the present invention relates to a method of monitoring the effects of environmental changes (e.g., diet) and/or genetic background on thermogenesis in various organisms (animals, plants, tissues, and cells).
• the method can comprise: i) measuring heat produced either by an organism, using infrared thermography, under different environmental conditions (e.g., fed different diets: high or low fat, protein, or carbohydrate diets) or by organisms with different genetic backgrounds (e.g., inbred animals, populations), ii) exposing the organism(s) to various agents (e.g., placebos or thermogenic agents; including untreated controls), iii) measuring the heat produced by the organism(s) after treatment with the agent using infrared thermography, iv) comparing the measurements in steps (i) and (iii), to determine the influence of environmental changes and genetic background.
• agents e.g., placebos or thermogenic agents; including untreated controls
• the present method can be used to identify, predict, characterize, rank, and select how different environments (e.g. diet) or genotypes can influence basal or agent-induced thermogenesis.
• environments e.g. diet
• genotypes can influence basal or agent-induced thermogenesis.
• the organisms can be naturally occurring (e.g., house mouse), inbred (e.g., AKR/J mice), or engineered (e.g., transgenic mice).
• the method can comprise measuring the heat produced using infrared thermography upon changing the diet, circadian cycle, diurnal rhythm, altitude, barometric pressure, humidity, temperature, noise, sleep status, physical or mental stress and injury of the cell or organism.
• Diets can be poorly defined (e.g., cafeteria diets) or well characterized (e.g., laboratory chow). The organisms can be fed on scheduled diets or ad libitum.
• the agents that alter thermogenesis can be naturally occurring or synthetic, known or unknown, safe or toxic, and anabolic, catabolic, or without effect.
• Environmental (dietary or otherwise) changes, genotypes, or agents that enhance thermogenesis (body heat production) are potentially useful for identifying catabolic states.
• Environmental changes, genotypes, or agents that suppress thermogenesis (body heat production) are potentially useful for identifying anabolic states.
• the present invention relates to a method of monitoring drug-drug interactions in various organisms (humans, animals, plants, tissues, and cells).
• the method comprises: i) measuring the heat produced by the organism (cells, etc.), using infrared thermography, before exposure to the agent(s), ii) exposing the organism (cells, etc.) to a single agent and to multiple agents (e.g., by adding to culture medium or dosing by injection, gavage, topical application, etc.), iii) measuring the heat produced by the organism (cells, etc.) after treatment with a single agent and after treatment with multiple agents, using infrared thermography, iv) determining the differences in heat produced in steps (i) and (iii) and comparing the differences in heat produced after exposure to single agents with the heat produced after exposure to combined agents.
• thermogenic response A difference in the heat produced after exposure to multiple agents (as opposed to single agents) indicates that the agents interact or are eliciting a thermogenic response.
• agents that result in a change in thermogenesis when used in combination, relative to when used singly are proposed to be involved in pharmcodynamic drug-drug interactions. Such interactions can be potentially toxic or beneficial to the organism, tissue, or cells.
• infrared thermography can be used to identify, predict, characterize, rank, and/or select how different agents (e.g., drugs) interact with each other. There are no restrictions to the type and number of agents, cells, tissues, and organisms that can be used.
• the agents can be naturally occurring, synthetic, agonists, antagonists, inhibitors, activators, safe, toxic, anabolic, catabolic, known, or unknown.
• the cells, tissues, and organism can be derived from plants, animals (e.g., man), fungi, protozoans, or monera.
• Infrared thermography can be used to measure the heat produced by cells, tissues, and/or organisms upon changing various pharmacokinetic and pharmacodynamic parameters, including altering the duration of exposure, the concentration of agent(s), pharmaceutical compositions, and number of agents used.
• the present invention relates to a method of monitoring hair loss (alopecia) and regrowth.
• infrared thermography can be used to identify, predict, characterize, rank, and/or select how different treatments or environmental stimuli alter hair growth.
• the types of treatments can include diet, exercise, pharmacological, radioactive, or surgical intervention and can be invasive or noninvasive.
• the stimuli for altering hair growth can be present naturally in the environment (e.g., radon gas) or a result of environmental contamination (pollution, such as pesticides).
• infrared thermography can be used to monitor the safety, potency, and efficacy of various treatments (natural or artificial) on hair loss and growth.
• the present invention relates to a method of evaluating safety profiles of pharmacologic agents.
• various proteins e.g., cytochrome P450s etc.
• organelles e.g., microsomes, etc.
• cells, tissues, and organ types targeted by an agent can be isolated, treated with varying concentrations of the agent and heat production monitored using infrared thermography.
• This method can comprise: i) determining the potency and efficacy of an agent on stimulating or inhibiting heat production in the desired target (e.g., a protein, organelle, cell, tissue, or organ involved in the therapeutic effect of an agent), ii) determining the potency and efficacy of an agent on stimulating or inhibiting heat production in an undesirable target (e.g., a protein, organelle, cell, tissue, or organ involved in a toxic effect of an agent), iii) determining the selectivity of the agent by comparing the potency and efficacy in steps (i) and (ii).
• Pharmacological agents that show increased selectivity between the various targets e.g., protein, organelle, cell, tissue, and/or organ
• the effects of varying the concentration of the test agent on heat generated by binding-partners and/or enzyme catalysis can be used to evaluate the selectivity and safety profile against multiple targets.
• Optimum selectivity between desirable and undesirable targets e.g., cell types, binding-partners, or enzymes
• desirable and undesirable targets can be determined readily by one skilled in the art.
• the present invention relates to a method of evaluating the physical state and/or amount of a compound(s).
• the physical state of a compound can be determined using this method as it relates to a compound changing its physical properties of going from a solid (i.e. frozen liquid) to a liquid (i.e. melting), a liquid into a solid (i.e. crystallization), a liquid into a gas (i.e. evaporation, vaporization), a solid into a gas (i.e. sublimation).
• This embodiment can be applied but is not limited to compounds in open vessels, closed systems, pressurized vessels (i.e. inhalants).
• the amount of a liquid can be measured using the present invention. Consistent with this embodiment, each varying amount of the test agent generates a unique heat profile whereby the amount of agent present can be measured by its unique heat characteristics.
• the invention has applicability in connection with virtually any animal or animal tissue.
• mammals including primates (e.g. humans) and any of the commonly used laboratory animals (e.g., rats, mice, hamsters, guinea pigs and rabbits) as well as to birds, amphibians and reptiles and insects.
• Adipocytes Human subcutaneous adipocytes were purchased from Zen-Bio,
• C3H10T1/2 clone 8 fibroblasts were differentiated into adipocytes as previously described (Lenhard et al, Biochem. Pharmacol. 54:801-808 (1997), Paulik and Lenhard, Cell Tissue Res. 290:79-87 (1997)).
• triglyceride accumulation was determined by adding lipoprotein lipase and GPO-Trinder reagent (assay kit 337-B, Sigma Diagnostics, St. Louis, MO) to the cells (50 ⁇ l/ cm 2 ) and incubating the lysates at 37°C for 2 hours. The optical density was measured using a spectrophotometer set at a wavelength of 540 nm. Lipolysis was measured as previously described (Lenhard et al, Biochem. Pharmacol. 54:801-808 (1997)).
• Human skeletal muscle cDNA (#7175-1) was purchased from Clontech (Palo Alto, CA). UCP2 specific-sequences were PCR amplified from the sample using oligonucleotide primers that matched the 5' and 3' ends of a published sequence (GenBank U82819). Vent polymerase was used (New England Biolabs, Beverly, MA) in a standard reaction mixture with 2 mM MgSO 4 and 5% DMSO. The cycle parameters were 94°C for 1 min., 55°C for 1 min., and 72°C for 1 min., repeated 29 times. The sample was passed over an S-400 spin column (Pharmacia, Piscataway, NJ) prior to ligation in a vector for transformation of E.
• S-400 spin column Pharmacia, Piscataway, NJ
• the UCP2 coding sequence was amplified by PCR using primers with the sequence AAAAAACCCCGGATCGAATTCATGGTTGGGTTC AAGGCCA (SEQ ID NO: 1 ) (sense) and
• CATTGTTCCTTATTCAGTTACTCGAGTTAGAAGGGAGCCTCTCGGGA (SEQ ID NO:2) (antisense) followed by a second PCR using primers with the sequence TTAACGTCAAGGAGAAAAAACCCCGGATCG (SEQ ID NO:3) (sense) and GAAAGGAAAAACGTTCATTGTTCCTTATTCAG (SEQ ID NO:4) (antisense).
• the PCR product was cloned into pYX233 (R&D Systems) by homologous recombination in yeast strain W303 (a/a homozygous for ade2-1, his3-1, 15 leu2-3, 112, trp1-1, ura3-1). Yeast transformants were selected on BSM-trp agar (Bio 101 , Vista, CA). The correct UCP2 sequence was verified by sequencing plasmids back- extracted from yeast to E. coli.
• mice were housed 5 animals/cage at 72° F and 50% relative humidity with a 12 hr light and dark cycle, and fed chow diet (NIH R&M/Auto 6F-Ovals 5K67, PMI Feeds® Inc., Richmond, Indiana). Animals starting at 41 days of age were orally gavaged once daily (8:00- 9:00 AM) with 0.05M N-methylglucamine (control) and 5 mg/kg GW1929 in 0.05M N-methylglucamine. After 2 weeks of dosing, the animals were dosed (intraperitineal) with 1 mg/kg of CGP12177A, and the animals were anesthetized with isofluorane.
• Infrared thermographic images and temperature calculations were recorded using an Agema Thermovision 900 Infrared System .
• the data was calculated as the mean and standard error from experiments performed on > 6 animals per treatment group. This research complied with the principles of laboratory animal care (NIH publication No. 86-23, revised 1985) and company policy on the care and use of animals and related codes of practice.
• Male AKR/J, C57BL/6J, and SWR/J mice were purchased from 4-8 weeks of age from Jackson Laboratories (Bar Harbor, ME). Mice were fed high and low fats diets containing high sucrose as defined by Surwit et al., (Metabolism 44(5):645-651 (1995)).
• Heat generation was measured using a Stirling cooled Agema Thermovision 900 Infrared System AB (Marietta, GA) equipped with a SW Scanner and 40° x 25° lens which detects a 2-5.4 micron spectral response.
• the scanner had an internal calibration system with an accuracy of 0.08°C.
• the focal distance was 6 cm. Images were captured using a recurs function set at 16 or an averaging function set at 32.
• the data was analyzed using OS-9 advanced systems and ERIKA 2.00 software according to the manufacturer's specifications (FLIR Infrared Systems AB, Danderyd, Sweden).
• Thermography of adipocytes was performed by maintaining the ambient temperature of the cultured cells at 37 ⁇ 0.02°C using a Queue Systems Inc. (Parkersburg, W.V.) incubator, model QWJ500SABA. Spectral analysis of yeast was performed at 30 ⁇ 0.02°C using the same incubator system. After treating the cells with experimental agents (e.g., rotenone etc.), the temperature was equilibrated for 10 minutes in the incubator before measuring real time thermogenesis for all microtiter plate applications. Various color scales in the visible wavelength were used to depict the temperature fluctuations of the recorded images. Although temperature scales are constant, the color scale images are variable and can be adjusted with level and span controls.
• the outer wells of the culture plates were omitted from the detection system because increased thermal conductance occurred at the edge of the plates. Larger diameter wells (i.e., >1 cm) were less satisfactory because a meniscus effect was observed that resulted in uneven thermal conductance. Further, the amount of media per well was critical, since too much media decreased the signal and too little media created a meniscus resulting in increased background noise. The best results were obtained using 50 ⁇ l/well using both 96-well plates containing adherent adipocytes and 384-well plates containing yeast suspensions. Enclosure of the detection system and objects that were being profiled was essential for minimizing changes in temperature and reflectivity (i.e., thermal noise) which result from air currents and light, respectively.
• thermal noise i.e., thermal noise
• the methods of the present invention utilize an apparatus that, advantageously, consists of a high resolution infrared imaging system and a central processing unit with appropriate software for data analysis.
• An example of a suitable system is that produced by FLIR Infrared Systems (the AGEMA 900) or the non-contact infrared thermometer (C-1600MP) produced by Linear Laboratories.
• Figure 1 shows a schematic diagram of such an apparatus for infrared thermography of cell-free systems or cell culture.
• the isothermal chamber constructed from a non-reflective material that provides a heat sink to dampen out temperature fluctuations (e.g., anodized aluminum) minimizes thermal noise (e.g., reflection and air currents) from the culture plates and surrounding environment.
• a plate holder can be placed within the isothermal chamber to maintain thermal uniformity across the plate. Use of an incubator also prevents fluctuations in the surrounding temperatures and improves cellular responses and viability.
• the camera monitors real time heat production from the cells in culture with images recorded by a central processing unit for data capture and software analysis tools for further data analysis.
• Figure 2 shows a schematic diagram of an apparatus designed for infrared thermography of the intrascapular region of mice. This apparatus shares features with the apparatus in Figure 1 , including the infrared camera, isothermal chamber and computer interface.
• It can also include an infrared screening platform with anesthetic manifolds for maintaining the animais under anesthesia, a tightly regulated heating block, for example, made out of anodized aluminum, and an isothermal chamber to maintain air currents at a minimum.
• intrascapular brown adipose tissue IBAT
• IBAT intrascapular brown adipose tissue
• This tissue contains abundant mitochondria which express the anion transporter, uncoupling protein (UCP1 , formerly known as UCP;
• UCP1 uncouples oxidative phosphorylation from respiration in IBAT resulting in generation of heat instead of ATP.
• UCP1 is not abundant in Homo sapiens
• UCP2 (Fleury et al, Nat. Genet. 15:269-272 (1997)) is abundantly expressed in humans.
• UCP2 mRNA is ubiquitously expressed and its expression is altered in obesity (Enerback et al, Nature 387:90-94 (1997)).
• the antidiabetic thiazolidinediones e.g., troglitazone
• the nuclear receptor PPAR ⁇ e.g., PPAR ⁇
• UCP2 plays a critical role in regulating energy balance (Shimabukuro et al, Biochem. Biophys. Res. Commun. 237:359-361 (1997)).
• UCP1 regulates mitochondrial-mediated thermogenesis in rodents
• UCP2 plays a similar role.
• the UCP2 gene was cloned from a human cDNA library and expressed in yeast using a galactose-inducible expressiQn system.
• Fig. 4A expression of UCP2 in yeast resulted " in increased thermogenesis relative to cells lacking UCP2.
• treatment of the cells with rotenone inhibited UCP2-mediated thermogenesis (Figs. 4A and 4B).
• ⁇ 3 AR beta 3 adrenergic receptor receptor
• CHO Chinese Hamster Ovary
• the engineered CHO cells overexpressing the ⁇ 3 AR receptor were profiled thermally tor their responsiveness to the well characterized ⁇ -AR agonist, isoproterenol (Fig. 5).
• Fig. 5 CHO cells were responsive in a dose-dependent manner to isoproterenol, indicating infrared thermography can be used to evaluate, identify and rank order ligands for cell surface receptors (e.g. ⁇ 3 AR).
• thermography as a non-invasive tool that can be used to rank, select and identify compounds for drug discovery using engineered cell models overexpressing foreign proteins or can be extended to antisense expression.
• infrared imaging can be used to monitor the activity of intracellular kinase activity as exemplified by the dose-dependent response of CHO cells when administered the well-characterized Protein Kinase A (PKA) agonist.forskolin (Fig. 5).
• PKA Protein Kinase A
• Fig. 5 Protein Kinase A
• Troglitazone is an antidiabetic agent that increases anabolism (e.g., lipogenesis and mitochondrial mass) and decreases catabolism (e.g., basal lipolysis and aerobic respiration) in C3H10T1/2 cells (Lenhard et al, Biochem. Pharmacol. 54:801-808 (1997)).
• the effects of troglitazone on these cells is a result of activation of the transcription factor PPAR ⁇ /RXR which, in turn, induces differentiation of the stem cells into adipocytes (Lenhard et al, Biochem. Pharmacol.
• thermogenesis is suppressed as these cells differentiate into adipocytes.
• thermogenesis is not sufficient for stimulation of thermogenesis in adipocytes.
• This finding is in agreement with the suggestion that in addition to increased UCP expression other signals (e.g., ⁇ -AR stimulation) are needed to stimulate thermogenesis in adipocytes (Lenhard et al, Biochem. Pharmacol. 54:801-808 (1997), Paulik and Lenhard, Cell Tissue Res. 290:79-87 (1997)).
• ⁇ -AR stimulation e.g., ⁇ -AR stimulation
• infrared thermography can be used to study the pharmacological effects (i.e. efficacy, potency, kinetics, etc) of agents that affect cell growth and/or differentiation, such as troglitazone and other nuclear receptor ligands, on heat production.
• Reagents that activate the transcription factor PPAR ⁇ /RXR have pharmacological potential as antidiabetic agents.
• Infrared thermography using the method described herein can be used to monitor the effects of such drugs in animal model systems. This application has significant importance in drug development and testing.
• FIG. 8A shows the thermogenic effect of GW1929x treatment of a group of ob/ob mice. Control mice were treated in the same manner as the experimental group but with the drug vehicle lacking the drug. As expected, treatment with the antidiabetic agent for a period of two weeks prior to assay causes a reduction in IBAT thermogenesis in the treated mice compared to the control animals (Fig. 8A). This thermogenic assay in whole animals also has quantitative value for determining drug effectiveness.
• the minimal effective dose (MED) for a group of PPAR ⁇ agonists in whole animals correlates directly with the ability of the drug to suppress thermogenesis as detected by infrared thermography in cell culture (Figs. 8B and Fig. 7 described in Example 4).
• Catecholamines are postulated to regulate body temperature and composition (Blaak et al, Int. J. Obes. Relat. Metab. Disord. 17 Suppl
• the mitochondrial electron transport inhibitor, rotenone inhibited thermogenesis of cells treated with 50 nM of the various ⁇ -AR-agonists (Fig. 9).
• An inhibitor of protein synthesis, cycloheximide (100 ⁇ M) had no effect on ⁇ 3 -AR-mediated thermogenesis in these cells.
• thermogenesis was greater after 15 min than after 18 hours treatment with CL316243 (there was a dose dependent thermogenic effect of CL316243 (dose range used was 0.8-100nM)).
• ⁇ 3 -AR-induced thermogenesis may be an acute response that does not require increased protein (e.g., UCP) synthesis.
• UCP protein
• these results do not preclude a role for ⁇ 3 -AR in regulating UCP synthesis (Rehnmark et al, J. Biol. Chem. 265:16464-16471 (1990), Lafontan and Berlan, J. Lipid Res. 34:1057- 1091 (1993), Silva, Mol. Endocrinol. 2:706-713 (1988)).
• VEGF Vascular endothelial growth factor
• KDR kinase domain receptor
• MAPKKK mitogen-activated protein kinases
• p90rsk S6 kinase
• thermography was used to measure the heat generated by human epithelial vascular cells (HUVEC) cells treated with VEGF. As shown in Figure 10, VEGF induced thermogenesis in HUVEC cells, indicating infrared thermography may be used to monitor enzymatic reactions (e.g., kinase/phosphatase activity) in cell culture. Thus, infrared thermography can be used to evaluate the efficacy and potency of compounds on enzymatic reactions.
• HUVEC human epithelial vascular cells
• thermography As shown in Figure 11 , the mixing of 0.25 mM NaOH with varying concentrations of HCI exhibited a dose-dependent thermogenic response when measured by infrared thermography.
• ligand e.g., drug
• binding partners e.g., receptors
• Further examples include using infrared thermography to monitor drug-receptor, protein-protein, protein-DNA, DNA-DNA, DNA-RNA and protein-carbohydrate interactions.
• Infrared thermography as presented in this invention can also be used to measure thermogenesis in highly defined cell-free systems. Catalytic agents are often characterized while immobilized by attachment to inert solid surfaces such as combi-chem beads (Borman, Chem. Eng. News 74:37 (1996)). Thermal analysis of catalyst reactions on combi- chem beads was therefore tested using the present invention. Catalytically active combi-chem beads were analyzed while immersed in solvent in a 25 mL beaker or in a 96 well microtiter plate. Fig 12 shows that thermogenic output localized to the area of the beaker containing the beads (Fig. 12A, arrows) (0.3°C temperature difference) or to the wells of the microtiter plate that contain active but not inactive beads (Fig. 12B). Thus, infrared thermography can be applied to measure real-time catalyst activity in a non- invasive and non-destructive manner.
• Aerosol systems, metered dose inhalers (MDI), used in drug delivery are associated with decreased temperature in the apparatus chamber during delivery. This effect has significant implications for the efficiency of drug delivery.
• a decrease in chamber temperature often causes drug crystallization within the MDI chamber and stem, ultimately, resulting in inefficient delivery of the drug.
• Infrared thermal imaging can be used to monitor the temperature loss during drug delivery and to test modified devices that alleviate or ameliorate the problem.
• infrared thermography was tested as a measure of actuation-induced chamber cooling of a MDI.
• Figure 13A shows a thermal profile in real time of an MDI after 0,1 or 5 consecutive actuations.
• the thermal image was analyzed for temperature fluctuation in the three following areas: area 1 - the surface of valve stem/expansion chamber; area 2- the surface of the middle of the canister; area 3 - the canister head inside which sits the meteriricfchamber.
• the graphical representation of area temperature over time shown in Fig. 13B indicates that an actuation-dependent temperature decrease occurs specifically in the valve stem/expansion chamber. Iterative drops in temperature occur with each actuation.
• Assays available for measuring bioavailabilty of inhalants involve measuring the uptake of radiolabeled inhalant in selected tissues (eg. lung).
• Infrared thermography provides a non-invasive method for measuring the bioavailibilty/bioactivity of inhalant compounds.
• infrared thermography was used to measure the thermal activity induced by an inhalant in the thoracic area of nude mice.
• Figure 13 C shows a thermal profile of nude mice that were treated with inhalants containing either vehicle or albuterol.
• the graphical representation of the torso area temperature indicates that the torso temperature increases after 2.5 minutes of dosing with inhalant (Fig. 13D).
• Infrared Thermography Can Be Used to Measure IBAT Thermogenesis in Mice Treated with ⁇ 3 -AR-Agonists
• thermogenesis The ability of ⁇ 3 AR-agonists to stimulate thermogenesis in cultured adipocytes and CHO cells was shown and discussed in Examples 3 and 6 and Figures 5, 8 and 9 above. Since there are potential clinical applications for the use of catabolic agents (e.g., ⁇ 3 AR-agonists) in the treatment of diabetes and obesity, it is important to show that infrared thermography can measure ⁇ 3 AR-agonist-induced effects in whole animals.
• Figs. 14A and 14B show that infrared thermography can be used to measure a dose-dependent and time-dependent increase in the intrascapular brown adipose tissue region (IBAT) thermogenesis in animals challenged with a ⁇ 3 AR-agonist .
• IBAT intrascapular brown adipose tissue region
• thermogenesis ⁇ 3 AR agonist-induced thermogenesis reflects increased catabolic activity was tested by direct measurement of serum glycerol in treated animals.
• Monoamine reuptake inhibitors are a class of drugs that stimulate catabolic activity (Stock, Int. J. Obesity 21 :525-29 (1997)).
• the effect of a representative monoamine reuptake inhibitor, GW473559A was monitored by infrared thermography of treated mice.
• Figure 15 shows that infrared thermography measures a dose-dependent (Fig 15A) and a time- dependent (Fig. 15B) increase in IBAT thermogenesis in ob/ob mice treated with GW473559A.
• infrared thermography provides a non- invasive, sensitive, and robust surrogate assay for the bioavailability and activity of compounds.
• ⁇ 3 AR-agonists Treatment with ⁇ 3 AR-agonists to control diabetes or obesity can require therapy that spans extended time periods (weeks or months).
• One desired outcome of ⁇ 3 AR-agonist treatment can be weight loss.
• Data shown in Figure 16 demonstrate that infrared thermography can be used to predict weight loss resulting from drug treatment.
• AKR mice were placed on a high fat diet and treated with either placebo or ⁇ 3 AR agonist (twice daily) for 2 weeks.
• infrared thermography can provide a non-invasive surrogate assay for both preclinical and clinical use for compound selection, and evaluation of efficacy and potency.
• thermography could be used to measure the effects of diet on drug- induced .changes in heat production in animals with different genotypes.
• thermogenic response As shown in Figure 17, the obesity prone mice, AKR/J, had a greater thermogenic response to BRL37344 when fed the higher fat diet. In contrast, the obesity resistant mice, SWR/J, had a greater thermogenic response when fed the lower fat diet. There was little difference in the thermogenic response of C57BL/6J mice on a high or low fat diet.
• thermogenic output of the dorsal area of a human subject will vary as a function of the time of day and the subject's pattern of food intake. This is demonstrated in a profile of a patient whose dorsal temperature is monitored by infrared thermography at time points before and after a meal.
• Figure 18A shows quantitative analysis of the thermographic profile for time points before and after lunch.
• Figure 18B shows a graph summarizing similar measurements made in 2 male subjects and 1 female subject before and after lunch (Torso Delta T) on three separate occasions.
• This data set is consistent and reproducible, and indicates that infrared thermography of humans is possible for monitoring thermogenesis. This method may be useful to monitor many situations applicable to human patients, such as diet modulation, drug treatment, drug/drug and drug/environment interactions. Based on the results using infrared thermography, changes in diet and environment and drug use can be prescribed.
• the sympathomimetic agent ephedrine has been reported to have potent thermogenic and anti-obesity properties in rodents (Astrup et al, Am. J. Clin. Nutr. 42:183-94 (1985)).
• Weight loss and body composition measurements are markers primarily used to determine the efficacy of pharmolpgical treatments for obesity. However, studies utilizing these markers lend to be time consuming, large and costly. In order to circumvent these problems, surrogate markers have been developed. Indirect calorimetry is used to determine resting metabolic rate but due to its complexity, it is not widely used. Biochemical markers such as glucose, glycerol, nonesterified fatty acids, triglycerides have been used but are invasive. However, thermogenic imaging has never been used to measure the properties of ephedrine in humans.
• thermogenesis The effect of ephedrine on thermogenesis in two human subjects was detected by infrared imaging 60 minutes after treatment with ephedrine at a dose of 0.6-0.7 mg/kg.
• infrared thermography can be used as a non-invasive surrogate assay to evaluate the efficacy, potency, pharmacokinetics, pharmocodynamics of drugs in clinical studies.
• GW1929 is an agent that improves glycemic control in diabetic animals by activating transcriptional activity of the ligand-activated nuclear receptor PPAR ⁇ .
• CGP12177A is an anti-obesity agent that acts via stimulation of the cell-surface ⁇ 3 -adrenoceptor (Kenakin, Lenhard and Paulik, Curr Prot Pharm; 1(unit 4.6):1-36 (1998)). Since many diabetic patients are obese, it was of interest to determine if these two agents (i.e., GW1929 and CGP12177A) had any pharmacodynamic interactions. Thus, db/db mice were treated for 2 weeks with or without GW1929.
• VEGF Vascular endothelial growth factor
• Nude mice were injected with either VEGF peptide or a control followed by themographic imaging of both injection sites.
• Figure 21 shows thermographic images that demonstrate enhanced thermogenesis in the local area of the VEGF injection, but not in the local area of the control injection.
• infrared imaging can be used to monitor the effects of agents that alter tissue vascularization.
• Tumor temperature is an indicator of the metabolic rate in the tumor. Tumors are dependent on the presence of VEGF for maximum metabolic activity and tumor temperature reflects changes in the availability of VEGF. This relationship is demonstrated by thermographic analysis of tumor-bearing mice treated with either an anti-VEGF antibody or a nonspecific anti-lgG antibody. Figure 22 shows quantitative thermographic analysis demonstrating that tumor temperature decreases when VEGF is neutralized by the presence of anti-VEGF antibody.
• infrared imaging can be used to monitor the effects of anti-cancer therapies and as an aid in anti-cancer drug development.
• Hair loss can result from undesirable side-effects of various therapies (e.g., radiation treatment of cancer patients, surgery etc.) and can occur naturally with age, whereas surgical or pharmacological intervention can restore hair growth. Since hair provides insulation against heat loss and aids in the maintenance of a constant body temperature, it was of interest to determine if infrared thermography can be used to measure hair loss. The lack of progress in the treatment and prevention of chemotherapy-induced alopecia is in part due to the lack of a reproducible animal model as well as a quantitative method to measure hair-loss.
• Fig. 23 shows thermal images (Fig. 23A) and quantitative analysis (Fig. 23B) demonstrating increased thermal activity in both the fronts and backs as a result of hair-loss.
• MED Male erectile dysfunction
• Increased local thermogenesis is associated with increased local blood flow.
• One drug that acts in this manner and treats MED is Pinacidil.
• Figure 24 shows that infrared thermography detects a Pinacidil-induced increase in thermogenesis in the genitalia of rats 2 hours after dosing with either 3.0 or 0.3 mg Pinacidil /kg.
• infrared thermography provides a quantitative and non- invasive method for identifying and evaluating drug candidates for the MED indication as well as identifying candidates that can cause erection as a side effect.
• Arthritis is a disease characterized by inflammation of the joints and can be treated with antiinflammatory agents. Because inflammatory responses are associated with increased thermogenesis at the site of the response, arthritis and the efficacy of arthritis drugs can be monitored by thermography.
• an arthritis model was established by injecting one limb of a normal animal with peptidoglycan polysaccharide (PGPS) for two weeks. The other limb was not treated.
• PGPS peptidoglycan polysaccharide
• Infrared thermography on both limbs after the arthritis-inducing treatment demonstrates a higher level of thermogenesis in the treated limb than in the untreated limb ( Figure 25A) indicating infrared thermography can be used to monitor the ability of agents to cause inflammation.
• thermography is a useful tool for monitoring inflammatory responses and the efficacy of antiinflammatory agents as well as providing a method for screening, selecting and evaluating the effectiveness of drug candidates for treating arthritic indications.

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