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A novel approach for non-invasive diagnostic imaging of invasive fungal infections

A novel approach for non-invasive diagnostic imaging of invasive fungal infections. Fungal Infections. Invasive fungal infections (IFI) are a serious threat to human health mostly due to high proliferation rate of fungal pathogens.

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A novel approach for non-invasive diagnostic imaging of invasive fungal infections

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  1. A novel approach for non-invasive diagnostic imaging of invasive fungal infections

  2. Fungal Infections Invasive fungal infections (IFI) are a serious threat to human health mostly due to high proliferation rate of fungal pathogens. These infections are especially dangerous in case of immunocompromised Individuals, whose number is growing due to: General aging of the population. Common use of immunosuppressive agents in patients who received transplants, implants, intensive anticancer therapies, or suffer autoimmune diseases, like rheumatoid arthritis or multiple sclerosis. Spread of immunocompromising diseases (e.g. diabetes, AIDS) www.gaffi.org www.life-worldwide.org

  3. The size of the fungal problem Over 300 million people affected by seriousFungal Infection worldwide 25 million - life-threatening and/or hugely debilitating www.fungalinfectiontrust.org

  4. Fungal Infections cases Eye infections Skin infections Lung infections Respiratory infections

  5. Annual estimated mortality from serious fungal infection worldwide The treat of fungal infections is not yet widely recognized, even though these infections are associated with high mortality rates. Over 1,350,000people are estimated to die annually

  6. Acute invasive fungal infections (IFIs) due to Candida species have a high associated mortality The most clinically significant fungal species are Candida and Aspergillus Candida spp. Crude Mortality Wisplinghoff et al. 2004 CID 39(3):309-17

  7. Challenges in IFI diagnostics The diagnosis of IFI is complex since it requires a combination of clinical observation, laboratory investigation, and radiological or other diagnostic imaging methods The clinical representation is often non-specific and can be caused by a wide range of infectious organisms, underlying illness or complications of treatment Uncertainties and controversies in disease definition and in selecting of the standardized methods for establishing the diagnosis

  8. Existing diagnostic approaches for IFI

  9. X-ray image of fungal lung infection This chest x-ray shows the affects of a fungal infection, coccidioidomycosis. In the middle of the left lung (seen on the right side of the picture) there are multiple, thin-walled cavities (seen as light areas) with a diameter of 2 to 4 centimeters. To the side of these light areas are patchy light areas with irregular and poorly defined borders. Other diseases that may explain these x-ray findings include lung abscesses, chronic pulmonary tuberculosis, chronic pulmonary histoplasmosis, and others Missed diagnosis often leads to death, serious chronic illness or blindness in, case of eye infections

  10. Early diagnosis saves lives Relationship between hospital mortality and the timing of antifungal treatment. Morell et al. AAC. 2005 49: 3640

  11. Our goal To save more lives by Development of fungal-specific probes, that could be used to reliably and promptly image pathogens in human body

  12. drug drug Our approach Diagnostic probe fluorophore fluorophore fluorophore fluorophore drug drug drug Incubation drug Detection Target Fungal cell Fungal cell Our approach takes advantage of highly specific and sensitive antifungal drugs as targeting molecules By coupling a fluorophore that can be visualized, the new probes would be able to detect the presence of fungal pathogens

  13. Antifungal drugs Antifungal drugs work by exploiting the differences between mammalian and fungal cells. Unlike bacteria both fungi and humans are eukaryotes, and therefore are more similar at the biological level. This makes it more difficult to discover drugs that target fungi without affecting human cells. As a consequence, many antifungal drugs cost severe side effects Therefore, for development of the diagnostic probes the ideal would be the drugs that do not have any interaction with host cells and tissues and target exclusively fungal cells. Also these drugs should have broad specificity targeting many fungal species

  14. H2N O N N N N N H H H O O O O O H H H H H N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H O O O O O O O O O O O O O O H H H O O O N NH N N N N H H H H H H H H H O O O N N N N N N O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H O Me N N N N O H OH N F Me O N N F N Antifungal drugs chosen for diagnostic probes design Caspofungin Binds with high affinity and specificity to its fungal target glucan synthase Posaconazole Potent and selective inhibitor of fungal cytochrome p 450 lanosterol 14 demethylase Both drugs have broad spectrum activity and are fungi-specific since they target the enzymes absent in mammalian cells

  15. H2N O N N N N N H H H O O O O O H H H H H N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H Caspofungin O O O O O O O O O O O O O O H H H O O O N NH N N N N H H H H H H H H C C H H H O O O N N N N N N 3 3 O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H NH NH NH NH NH O O O N N O N N N N N H H H O O O O O H H H H H B F F N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H O O O O O O O O O O O O O O H H H O O O N N NH N N N H H H Caspofungin-BODIPY H H H H H H O O O N N N N N N O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H The synthesis of fluorescently labeled caspofungin Antifungal drugs should have suitable groups for the attachment of the label BODIPY-Fl-NHS

  16. H2N O N N N N N H H H O O O O O H H H H H N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H Caspofungin O O O O O O O O O O O O O O H H H O O O NH N N N N N H H H H H H H H C C H H H O O O N N N N N N 3 3 O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H NH NH NH NH NH O O O N N O N N N N N H H H O O O O O H H H H H B F F N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H O O O O O O O O O O O O O O H H H O O O N N NH N N N H H H Caspofungin-BODIPY H H H H H H O O O N N N N N N O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 H O O O O H H H Modified caspofungin probe retains high antifungal activity with C. albicans Labeling should not affect targeting properties of the drugs. MIC 50 nM MIC 80 nM

  17. NO2 O Me O O N N N N H O C C O N F NH –(CH2)4-NH2 Me O N N F N The synthesis of fluorescently labeled posaconazole O Me N N N N H O OH N F Me O Posaconazole (SCH65592) N N F Succinic anhydride N Me-Im, DMSO O Me O O N N N N H O C C O N F OH Me O N 4-nitrophenol N F DCC, THF N O Me O O N N N N H O C C O N F O Me O N N F 1,4-diaminobutane N Fluorescent tag C H BODIPY-FL-NHS 3 N O N Me O B O N N F N N O F H C H C 3 C O N F NH –(CH2)4-NH Me O O Pos-Fl N N F N

  18. Imaging of C. albicansusing fluorescent caspofungin probe Fluorescent Light transmission The fluorescence intensity was consistent with the existing data about the distribution pattern of glucan synthase throughout the cell features, which was indicative for the specificity of labeling

  19. The labeling is highly specific Light transmission Fluorescent Labeling was reduced with partially drug-resistant strain No labeling occurred with the fluorophore, or drug alone Pre-incubation with non-modified drug eliminated the labeling

  20. Fluorescent imaging of Aspergillus fumigatus by caspofungin fluorescent probe Fluorescence Light transmission

  21. Next challenge In vivo imaging

  22. Fluorescence Molecular Tomography (FMT) is powerful in vivo imaging approach drug drug Imaging of fluorescent material (e.g. labeled microbial pathogens) in body can be achieved through excitation by powerful light source and detection of the induced fluorescence. Moving the excitation source around a scanned object with simultaneous signal acquisition allows 3-D imaging of the area of interest. Diagnostic probe fluorophore fluorophore drug drug Target fluorophore fluorophore drug drug Target Target Steps: 1. Probe injection 2. Clearance of unbound probe 3. Fluorescence imaging 4. Optical imaging 5. Superposition of fluorescent and optical images FMT requires near-infrared fluorescent labels

  23. Body tissues are transparent for near infrared light 630-760 nm Transparency window Transparency window of body tissues of body tissues window window

  24. The most of the current Near-Infrared fluorescent labels are bulky DDAO Cl Cl O O HO Cl Cl N N Cy 5 Cy 7.5

  25. Improving of the DDAO fluorescent properties by 7-amino modification DDAO Original pH-dependent emission ionizable group 48,000 Molar absorbance 3,600 Brightness 660 nm Emission maximum 7-amino DDAO Cl Cl Developed O R-HN compound pH-independent emission Cl Cl N N 53,000 Molar absorbance 6,000 Brightness 680 nm Emission maximum

  26. Light absorption spectrum of body tissues and the emission properties of the synthesized DDAO fluorophore 7-amino DDAO 7-amino DDAO emission is in the middle of tissue transparency window 7-amino DDAO is chemically and photochemically stable, which reduces photo bleaching

  27. Cl O HO N Cl Cl Cl The synthesis of DDAO-based reactive compounds for design of diagnostic probes Cl O S=C=N – – – (CH (CH (CH ) ) ) - - - NH NH NH 2 2 2 4 4 4 Amine-reactive derivative N Cl Cl Cl III 1. CSIm2 2. TFA 1,4-DAB-(AcOH)2 Cl Cl AcBr-PNP O H H H N N N – – – (CH (CH (CH ) ) ) - - - NH NH NH BrAc-NH O 2 2 2 2 2 2 4 4 4 – – – (CH (CH (CH ) ) ) - - - NH NH NH 2 2 2 4 4 4 N Cl Cl Cl DDAO II N Cl Cl Cl IV Inexpensive, commercially available Thiol-reactive derivative 3MI-Pr-OSu O O Cl O – – – (CH (CH (CH ) ) ) - - - NH NH NH N (CH2)2 NH C 2 2 2 4 4 4 O N Cl Cl Cl V Thiol-reactive derivative

  28. H2N O N N N N N H H H O O O O O H H H H H N N N H H H H H H H H O O O O O 2 2 2 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H H H H H H C C C C C N N N N N N N N 3 3 3 3 3 O O O O O O H H H H H H Caspofungin O O O O O O O O O O O O O O H H H O O O NH N N N N N H H H H H H H H H O O O N N N N N N O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H The synthesis of DDAO-labeled caspofungin DDAO-NCS H2N O N N N N N H H H O O O O O H H H H H S Cl O N N N H H H H H H H H O O O O O – – NH – (CH ) - NH C 2 4 C C C C C C H H H H H H C C C C C C H H H H H H N N N 3 3 3 3 3 3 3 3 3 3 3 H H H H H H H H Cl N H H H H H C C C C C N N N N N N N N 3 3 3 3 3 DDAO O O O O O O H H H H H H O O O O O O O O O O O O O O H H H O O O N NH N N N N H H H Caspofungin-DDAO H H H H H H O O O N N N N N N O O O C C C H H H O O O O O O H H H H H H O O O O O O O O O O O O O O O O O H H H H H H H H H H H 3 3 3 O O O O H H H H

  29. FMT detection of C. albicansin infectedmice kidneys using caspofungin-DDAO fluorescent probe treated non-treated infected non-infected

  30. Tomographic detection of C. albicans in live mice using fluorescent Casp-DDAO compound

  31. Imaging of the infection progress Day 0 Day 1.5 Day 2.5 Time elapsed after infection Signal 0.04 0.32 0.41 intensity

  32. Conclusions We developed fluorescently labeled fungi-specific diagnostic probes that can be used for microscopic and tomographic imaging of the pathogens. Tomographic imaging will allow more precise and targeted drug delivery, which could increase the therapeutic efficiency and as such, restrict the emergence of drug resistance

  33. Our team RHRI Center, Rutgers, the State University of New Jersey. USA Arkady Mustaev Lab David Perlin Lab Validation of the probes using Synthesis and characterization of the diagnostic probes Fluorescent microscopy and FMT David Perlin Arkady Mustaev Laura Wirpsza Min Hee Lee Yanan Zhao Shyamala Pillai Steven Park

  34. Stacking interactions Posaconazole-DDAO can act as “signaling” molecule, thereby providing high imaging contrast Quencher fluoropohore

  35. Incidence of Invasive Fungal Infections (IFI) Solid Organ Transplant 5 - 42% Kidney 5 – 14% Heart 5 – 32% Heart-Lung/Lung 15 – 36% Pancreas 18 – 38% Liver 7 – 42 % Bone Marrow Transplant 15 - 25% Intensive Care Unit 17% Singh, N. CID 2000; 31:545-53 Vincent JL. Intensive Care Med 1998; 24: 206-216

  36. Opportunistic Candida infections in the immunocompromised are a major cause of clinical morbidity and mortality, and are the 4th leading cause of nosocomial bloodstream infections Wisplinghoff et al. 2004 CID 39:309-17

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