Chronic inflammatory diseases affect millions of people daily. Patients undergo treatment of inflammatory diseases with varied results and many have a recurrence of locoregional or systemic disease throughout their lifetime. Despite major advances in the immunology field over the last decade, there are hurdles to overcome in the field, including, for example, improved patient quality of life by effective and preferably non-surgical ways of target-specific identification of the disease(s).
While the immune system normally provides a line of defense against foreign pathogens, there are many instances where the immune response itself is involved in the progression of disease. Diseases caused or worsened by a person's own immune response are autoimmune diseases such as multiple sclerosis, lupus erythematosus, psoriasis, pulmonary fibrosis, and rheumatoid arthritis and diseases in which the immune response contributes to pathogenesis such as atherosclerosis, inflammatory diseases, osteomyelitis, ulcerative colitis, Crohn's disease, and graft versus host disease (GVHD) often resulting in organ transplant rejection. Additional exemplary disease states include fibromyalgia, osteoarthritis, sarcoidosis, systemic sclerosis, Sjogren's syndrome, inflammations of the skin (e.g., psoriasis), glomerulonephritis, proliferative retinopathy, restenosis, and chronic inflammations.
Activated inflammatory cells, such as macrophages, can contribute to the pathophysiology of disease in some instances. Activated inflammatory cells can nonspecifically engulf and kill foreign pathogens within the cells by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins can be displayed on the inflammatory cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Inflammatory cell types that may be associated with inflammatory disease states include macrophages, monocytes, and progenitor cells, including endothelial progenitor cells.
The folate receptor (FR) is a 38 KDa GPI-anchored protein that binds the vitamin folic acid with high affinity (<1 nM). Following receptor binding, rapid endocytosis delivers the vitamin into the cell, where it is unloaded in an endosomal compartment at low pH. Importantly, covalent conjugation of small molecules, proteins, and even liposomes to folic acid does not alter the vitamin's ability to bind the folate receptor, and therefore, folate-drug conjugates can readily enter cells by receptor-mediated endocytosis.
Because most cells use an unrelated reduced folate carrier (RFC) to acquire the necessary folic acid, expression of the folate receptor is restricted to a few cell types. With the exception of kidney and placenta, normal tissues express low or nondetectable levels of FR. Folate binds to its cognate receptor (major isoforms: FR-α, FR-β, FR-γ and FR-δ) with high affinity (Kd<0.5 nM) and specificity. FR-α, overexpressed in 80% of all malignant cell types (e.g., ovarian, lung, breast, kidney) and FR-β, expressed in unique subsets of activated macrophages and certain hematological malignancies, constitute the two primary FR isoforms exploited for delivery of targeted therapeutic and imaging payloads. It has also been reported that FR-β, the nonepithelial isoform of the folate receptor, is expressed on activated (but not resting) synovial macrophages.
A folate-targeted single-photon emission computed tomography (SPECT) imaging agent (EC20) has been recently evaluated by Mary Jo Turk et al. (Arthritis & Rheumatism, Vol. 46, No. 7, July 2002, pp 1947-1955) to determine whether overexpression of a high-affinity folate receptor beta (FR-β) on activated macrophages can be exploited to selectively target imaging agents to sites of inflammation in rats with adjuvant-induced arthritis (AIA). Preclinical studies with such radio imaging agents emphasized the value of imaging arthritic tissues in vivo. The results suggest that it may also be useful for assaying the participation of activated macrophages in inflammatory processes such as rheumatoid arthritis. The same radio imaging agent (EC20) was more recently used to image atherosclerosis by audioradiography in apoliprotein E knockout mice by Wilfredo Ayalo Lopez et al. (The Journal of Nuclear Medicine, Vol. 51, No. 5, May 2010, pp 768-774) for the early detection of heart disease. However, some of the current radio imaging agents are not readily available and expensive, are difficult to model may lack the sensitivity for receptor-based imaging or may have undesirable half-lives or relatively short relaxation times.
Additional studies (Chrystal M Paulos et al., Arthritis Research & Therapy, 2006, 8:R77) have shown that folate-linked radiopharmaceuticals concentrate in arthritic joints, enabling visualization of such tissues by gamma scintigraphy. Selective removal of activated macrophages with folate-linked drugs could be exploited to treat Rheumatoid Arthritis with little toxicity to other tissues.
Immunotherapy treatment studies of another inflammatory disease, systemic lupus erythematosis (SLE), have also previously been performed where activated macrophages have been targeted with known folate-linked hapten conjugates (Bindu Varghese et al., Molecular Pharmaceutics, 4(5):679-85 2007).
EC-20 has also been used to detect FR+ macrophages accumulated at sites of infectious disease by gamma scintigraphic imaging of bacterial infection foci after infecting BALB/c mice with Staphylococcus aureus 
EC20 also has been used in the clinic to evaluate the RA joints in RA patients (Assessment of disease activity in rheumatoid arthritis using a novel folate targeted radiopharmaceutical Folatescan. Matteson E L, Lowe V J, Prendergast F G, Crowson C S, Moder K G, Morgenstern D E, Messmann R A, Low P S. Clin Exp Rheumatol. 2009 March-April; 27(2):253-9.)
Although SPECT scans are inexpensive, SPECT scans provide low resolution images when compared with PET scans. Therefore, multiple folate-targeted PET imaging agents are being evaluated in the preclinical stage, including several fluorine-18-labeled agents. However, the radionuclides used in PET imaging could possess either potential dosimetry issues due to their long half-lives [e.g. 86Y (t1/2˜14.7 h), 64Cu (t1/2˜12.7 h), and 66Ga (t1/2˜9.49 h)] or their requirement for on-site production due to their short half-lives [e.g. 11C (t1/2˜22 minutes), 13N (t1/2˜10 min), and 15O (t1/2˜2 min)]. Thus, major deficiencies exist in radioimaging.
Since radio imaging agents can damage the DNA that can lead to cancer, use of multiple doses of radio imaging agents are undesirable when monitoring response to therapy. Another disadvantage of radio imaging agents is that the patient has to stay in the hospital until the radio imaging agent clears through the body. Moreover, radioimaging agents can be expensive, may require on-site production, may not be able to store due to half-lives of the radionuclides. Therefore, there is a high medical demand to develop better and safer modalities for detection. In this regard, optical imaging offers many advantages over radio imaging, for instance, ease of synthesis, high purity, long term stability during storage, stability during the preparation, and a reasonable procedure for its synthesis and purification. Moreover, optical imaging agents are safe in clinical use.
Conventional fluorescent techniques use probes in the visible light spectrum (˜400-600 nm). Such a wavelength is not optimal to image inflammatory diseases as it is associated with a relatively high level of nonspecific background interference due to collagen in the tissues. Hence the signal to noise ratio from these conventional compounds is low. Moreover, the absorption of visible light by biological chromophores, in particular hemoglobin, limits the penetration depth of the fluorescent image penetration to a few millimeters. Thus disease sites that are buried deeper than a few millimeters in the tissue often remain undetected.
The combination of light absorption by hemoglobin in the visible light spectrum (<600 nm) and water and lipids in the IR range (>900 nm), offers an optical imaging window from approximately 650-900 nm in which the absorption coefficient of tissue is at a minimum. A suitable alternative to dyes that emit light in the visible range would be to develop dyes that can be used in the near infra red (NIR) range because light in the near infrared region induces very little autofluorescence and permeates tissue much more efficiently. Another benefit to near-IR fluorescent technology is that the background from the scattered light from the excitation source is greatly reduced since the scattering intensity is proportional to the inverse fourth power of the wavelength. Low background fluorescence is necessary for highly sensitive detection. Furthermore, the optically transparent window in the near-IR region (650 nm to 900 nm) in biological tissue makes NIR fluorescence a valuable technology for in vivo imaging and subcellular detection applications that require the transmission of light through biological components.
While the use of light in the NIR range for deeper tissue imaging is preferable to light in the visible spectrum, the NIR imaging dyes currently used in the art suffer from a number of challenges and disadvantages. These include a susceptibility to photobleaching, poor chemical stability, absorbance and emission spectra that fall within the same range as many physiological molecules (resulting in high background signal and autofluorescence). Moreover, most of the NIR dyes are not stable during the synthesis, not least due to the fact that conjugating to a ligand with an amine linker leads to multiple unwanted side products. Therefore, taking ligand-targeted NIR imaging agent for clinic can be expensive. Thus, current imaging methods that utilize NIR fluorescent probes for treatment of inflammatory diseases suffer from several drawbacks including being ineffective for deep tissue imaging (>5 mm from the surface); ineffective in quantifying fluorescence signal in mammalian tissues, and in production cost that increase preclinical-to-clinical translational time.
Progress has been made in using optical imaging agents that fluoresce in the near infrared spectral range. Previous advances have also included using non-targeted dyes as optimal contrast agents in the NIR-fluorescence spectral range of about 650 to about 1000 nm to identify inflammatory diseases, including rheumatoid arthritis. This included using cyanine dyes of the class of indotricarbocyanides, such as indocyanine green (ICG) known for its effectiveness in cardiovascular diagnostics and hepatic functional tests. While non-targeted fluorescent dyes have been shown to passively accumulate in some tissues, the resulting tissue-to-background ratios are often poor and the boundaries between inflammatory site and healthy tissues can be difficult to define.
Current research in NIR-fluorescence has led to new approaches for targeted imaging of inflammatory disease. Protease-activated NIRF probes have been studied in vivo for their ability to image the presence of collagen-induced arthritis in joints by targeting a particular cleavage site and resulting in fluorescence activation (Andreas Wunder et al., Arthritis & Rheumatism, 50(8):2459-2465 (2004)). Additionally, a recently developed NIR2-folate conjugate was tested for in vivo imaging of arthritis by targeting an abundance of folate receptors on activated macrophages in inflamed cells (Wei-Tsung Chen et al., Arthritis Research & Therapy, 7(2):R310-R317 (2005)).
However, NIR fluorescent dye-linker-ligand conjugate that comprise alternative folic acid derivatives as ligands with linkers to make better and brighter dyes have not been investigated for targeting inflammatory diseased tissue.
Thus, there remains a need for the use of alternative folic acid derivative-based-dye substances that can be used to specifically target inflammatory-diseased states and that have increased stability and brightness for use in vivo for imaging.