Patent Application
20230065522
This invention relates generally to the use of imaging probes for the diagnosis and treatment of cancer. In particular embodiments, the invention relates to a method for the non-invasive screening of a subject for oral squamous cell carcinoma (OSCC).
Poly(ADP-ribose)polymerase 1 (PARP1), an enzyme that is activated to repair damaged DNA in the cells, is increased in cancer cells due to the higher genomic instability. For certain subtypes of cancer, inhibition of PARP1 and the associated perturbation of the DNA repair pathway have been shown to be a successful chemotherapeutic treatment regime, both in preclinical as well as clinical research settings. Because PARP1 is overexpressed in various forms of cancer, it has become a high-value target for the treatment, diagnosis, and staging of cancer. It appears that no one has demonstrated highly elevated PARP1 expression in human oral cancer specimens before.
Oral cancer is defined as a malignant neoplasm on the lip or in the mouth, affecting more than 40,000 patients in the United States in 2014. Of all malignant oral cancers, oral squamous cell carcinoma (OSCC) is by far the most common epithelial malignancy in the oral cavity, accounting for over 90% of all cases. While the disease is not as threatening as other types of cancer when detected early (83% 5-year survival for local disease), nearly half of all patients display distant metastases at the time of diagnosis due to the lack of accurate screening protocols and screening tools for this type of disease.
Currently, visual inspection of suspicious lesions is the most common way of diagnosing OSCC. Besides tissue sampling, different optical imaging techniques can be used to non-invasively survey the mucosal tissue (optical or fluorescent imaging) and to obtain a more accurate picture of oral cancer growth, its location, spread and number of diseased lesions. Optical methods like chemiluminescence, which examines the higher density of nuclei in malignant tissues; tissue fluorescence, which measures the higher autofluorescence of cancerous lesions due to higher chromatin/metabolite content and stromal/collagen changes; or the imaging of toluidine blue, a dark-blue stain that binds to the DNA of cells, that accumulates to a higher degree in malignant tissues; have been used to non-invasively determine the presence of oral cancer. However, these tools do not target a specific biomarker and lack specificity, resulting in either high false-positive or false negative rates. Low specificity particularly hampers the detection of small or precancerous lesions, where accurate detection would have the highest impact.
The most abundant radiotracer used in the clinic today is 18F-FDG, a glucose analog with high uptake in most types of cancer. However, the use of 18F-FDG requires significant infrastructure (e.g. tomography scanners, availability of the short-lived 18F radioisotope, close proximity of a medical cyclotron, specialized personnel, etc.). Furthermore, the administration of radioisotopes is always linked to radioactivity absorbed by both patient and healthcare professionals. Thus, radiolabeled imaging agents are not suitable for screening purposes, and only patients with suspected or confirmed disease are typically subjected to PET scans. In the case of oral cancer, a screening-tool for early detection is needed to improve patient outcome. Furthermore, a bimodal imaging agent for dual use in screening and PET scanning would be optimal to improve outcomes of oral cancer.
There is a need for a technology that can detect cancer cell populations and treat tissues that are still viable from one or more successfully ablated tumor(s). Moreover, there is a clinical need to develop a robust and reliable imaging agent for oral cancer in the oral cavity. This technology must be able to detect malignant growth of the mucosa while still local and treatable.
Presented herein is a method for the non-invasive screening of a subject for oral squamous cell carcinoma (OSCC). Methods described herein use poly(ADP-ribose)polymerase 1 (PARP1), a targeted small molecule imaging agent, as a diagnostic tool to identify oral squamous cell carcinoma (OSCC) and improve surgical removal of tumors by intraoperative imaging. PARPi-fl can be used to detect malignant growth in the oral cavity, e.g., in a dentist's office setup, using a macroscopic fluorescence scanning imaging device after topical application of PARPi-fl, which preferentially accumulates in areas of elevated PARP1 expression. Furthermore, the employment of a microscopic device, such as a fluorescence endoscope or a handheld confocal microscope can improve identification of tumor margins and residual tumor tissue during tumor removal surgery.
In one aspect, the invention is directed to a method for the non-invasive screening of a subject for oral squamous cell carcinoma (OSCC), the method comprising the steps of: administering a composition comprising PARPi-fl onto and/or into tissue in an oral cavity of the subject, wherein fl comprises a fluorophore; flushing the tissue in the oral cavity of the subject to reduce or remove unbound components of the composition while leaving bound components of the composition; and detecting fluorescence from the fluorophore after the administering and the flushing steps, thereby identifying potential OSCC.
In certain embodiments, the subject is a human patient.
In certain embodiments, the method takes place in a dentist office or other non-surgical setting.
In certain embodiments, the administering is topically administered.
In certain embodiments, the composition is a liquid composition.
In certain embodiments, the liquid composition is selected from the group consisting of an oral rinse, a mouthwash, a spray, an intravenous injection, and a local intramuscular injection.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the flushing comprises waiting a period of time following the administering step such that the tissue clears unbound components of the composition (e.g., via the lymphatic system, endocytosis).
In certain embodiments, the flushing comprises rinsing (e.g., with water or saline) and/or gargling (e.g., with water or saline).
In certain embodiments, potential OSCC is identified at the area of accumulation. In certain embodiments, potential OSCC is identified using a fluorescence scanning imaging device. In certain embodiments, potential OSCC is identified following exposure of the oral cavity tissue to excitation light.
In another aspect, the invention is directed to a method for intraoperative detection of a tumor margin and/or residual tumor tissue during tumor removal surgery, the method comprising the steps of: administering a composition comprising PARPi-fl onto and/or into a viewable tissue surface of the subject, wherein fl comprises a fluorophore; flushing the viewable tissue surface to reduce or remove unbound components of the composition while leaving bound components of the composition; and detecting fluorescence from the fluorophore after administration to the viewable tissue surface, thereby identifying the tumor margin and/or residual tumor tissue.
In certain embodiments, the tissue is from cancers of the aerodigestive tract, gastrointestinal tract, urinary tract, ovarian cancer, oral cancer, colorectal cancer, stomach cancer, bladder cancer, cervical cancer, retinal cancer, skin cancer, lung cancer, bronchus cancer, esophageal cancer, or any cancer that can be observed close to the tissue surface with a laparoscopic microscope (e.g., pancreatic, liver, kidney, spleen) or any cancer that is surgically resected, and where tissue margins can be observed (e.g., brain).
In certain embodiments, the administering is topically administered or intravenously administered.
In certain embodiments, the viewable tissue surface is viewable to a surgeon.
In certain embodiments, the viewable tissue surface is viewable by direct exposure or by a camera with access to the tissue surface (e.g., via endoscope).
In certain embodiments, the composition is a liquid composition. In certain embodiments, the liquid composition comprises a rinse.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety has a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the flushing comprises waiting a period of time following the administering step such that the tissue clears unbound components of the composition (e.g., via the lymphatic system, endocytosis).
In certain embodiments, the flushing comprises rinsing (e.g., with water or saline) and/or gargling (e.g., with water or saline).
In certain embodiments, the tumor margin is identified using a fluorescence scanning imaging device.
In certain embodiments, the tumor margin is identified following exposure of the viewable tissue surface to excitation light.
In another aspect, the invention is directed to a composition comprising: a PARP inhibitor conjugated to a fluorophore.
In certain embodiments, the PARP inhibitor is selected from the group consisting of AZD2281, AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304, GPI15427, GPI16539, M 4827, NU1025, NU1064, NU1085, PD128763, PARP Inhibitor H (INH2BP), PARP Inhibitor m (DPQ), PARP Inhibitor VHI (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the composition is a liquid composition.
In certain embodiments, the liquid composition is selected from the group consisting of an oral rinse, a mouthwash, a spray, an intravenous injection, and a local intramuscular injection.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In another aspect, the invention is directed to a method of assessing efficacy of a cancer therapy in a subject receiving treatment for an oral carcinoma, the method comprising administering to the subject the composition.
In certain embodiments, the cancer therapy comprises chemotherapy, radiation, or surgery.
In certain embodiments, the administering occurs subsequent to the cancer therapy.
In certain embodiments, the method further comprises administering a composition comprising 18F-PARPi to the subject.
In certain embodiments, the composition is in the same or in a different composition than the composition comprising PARPi-fl.
In certain embodiments, the administered composition enables PET imaging.
In certain embodiments, two orthogonal imaging modalities, PET for 18F-PARPi and optical imaging for PARPi-fl are conducted, thereby enabling screening (e.g., via optical imaging) and staging (e.g., via PET imaging) of disease.
In another aspect, the invention is directed to a composition comprising PARPi-fl, wherein fl comprises a fluorophore, for use in a method of in vivo diagnosis of oral squamous cell carcinoma (OSCC) in a subject, wherein the in vivo diagnosis comprises: delivering the composition onto and/or into tissue in an oral cavity of the subject; flushing the tissue in the oral cavity of the subject to reduce or remove unbound components of the composition while leaving bound components of the composition; and detecting fluorescence from the fluorophore after the administering and the flushing steps, thereby identifying potential OSCC.
In certain embodiments, the administering is topically administered.
In certain embodiments, the composition is a liquid composition. In certain embodiments, the liquid composition is selected from the group consisting of an oral rinse, a mouthwash, a spray, an intravenous injection, and a local intramuscular injection.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the flushing comprises waiting a period of time following the administering step such that the tissue clears unbound components of the composition (e.g., via the lymphatic system, endocytosis).
In certain embodiments, the flushing comprises rinsing (e.g., with water or saline) and/or gargling (e.g., with water or saline).
In certain embodiments, potential OSCC is identified at the area of accumulation. In certain embodiments, potential OSCC is identified using a fluorescence scanning imaging device. In certain embodiments, potential OSCC is identified following exposure of the oral cavity tissue to excitation light.
In another aspect, the invention is directed to a composition comprising PARPi-fl, wherein fl comprises a fluorophore, for use in an intraoperative method of in vivo diagnosis of a tumor margin and/or residual tumor tissue in a subject during tumor removal surgery, wherein the in vivo diagnosis comprises: delivering the composition onto and/or into a viewable tissue surface of the subject; flushing the viewable tissue surface to reduce or remove unbound components of the composition while leaving bound components of the composition; and detecting fluorescence from the fluorophore after administration to the viewable tissue surface, thereby identifying the tumor margin and/or residual tumor tissue.
In certain embodiments, the tissue is cancers of the aerodigestive tract, gastrointestinal tract, urinary tract, ovarian cancer, oral cancer, colorectal cancer, stomach cancer, bladder cancer, cervical cancer, retinal cancer, skin cancer, lung cancer, bronchus cancer, esophageal cancer, or any cancer that can be observed close to the tissue surface with a laparoscopic microscope (pancreatic, liver, kidney, spleen) or any cancer that is surgically resected, and where tissue margins can be observed (e.g., brain).
In certain embodiments, the administering is topically administered or intravenously administered.
In certain embodiments, the viewable tissue surface is viewable by direct exposure or by a camera with access to the tissue surface (e.g., via endoscope).
In certain embodiments, the composition is a liquid composition. In certain embodiments, the liquid composition comprises a rinse.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the flushing comprises waiting a period of time following the administering step such that the tissue clears unbound components of the composition (e.g., via the lymphatic system, endocytosis).
In certain embodiments, the flushing comprises rinsing (e.g., with water or saline) and/or gargling (e.g., with water or saline).
In certain embodiments, potential OSCC is identified using a fluorescence scanning imaging device.
In certain embodiments, potential OSCC is identified following exposure of the oral cavity tissue to excitation light.
In another aspect, the invention is directed to a composition comprising a PARP inhibitor conjugated to a fluorophore for use as an imaging agent.
In another aspect, the invention is directed to a composition comprising a PARP inhibitor conjugated to a fluorophore for use in in vivo diagnosis.
In certain embodiments, the PARP inhibitor is selected from the group consisting of AZD2281, AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304, GPI15427, GPI16539, M 4827, NU1025, NU1064, NU1085, PD128763, PARP Inhibitor H (INH2BP), PARP Inhibitor m (DPQ), PARP Inhibitor VHI (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the composition is a liquid composition. In certain embodiments, the liquid composition is selected from the group consisting of an oral rinse, a mouthwash, a spray, an intravenous injection, and a local intramuscular injection.
In certain embodiments, the composition is a gel, paste, or other solid or spray.
In certain embodiments, the PARPi-fl binds to PARP1 and preferentially accumulates (or only accumulates) in an area of elevated PARP1 expression, thereby signifying OSCC at the area of accumulation.
In another aspect, the invention is directed to a composition comprising a PARP inhibitor conjugated to a fluorophore for use in a method of assessing a cancer therapy in a subject, wherein the method comprises administering the composition to the subject.
In certain embodiments, the PARP inhibitor is selected from the group consisting of AZD2281, AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304, GPI15427, GPI16539, M 4827, NU1025, NU1064, NU1085, PD128763, PARP Inhibitor H (INH2BP), PARP Inhibitor m (DPQ), PARP Inhibitor VHI (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In another aspect, the invention is directed to a kit comprising: a PARP inhibitor conjugated to a fluorophore.
In certain embodiments, the PARP inhibitor is selected from the group consisting of AZD2281, AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304, GPI15427, GPI16539, M 4827, NU1025, NU1064, NU1085, PD128763, PARP Inhibitor H (INH2BP), PARP Inhibitor m (DPQ), PARP Inhibitor VHI (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A.
In certain embodiments, the fluorophore comprises a boron-dipyrromethene (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or a salt, moiety, or other equivalent thereof).
In certain embodiments, the fluorophore is conjugated to the PARPi or the fl and/or PARPi moiety(ies) are modified to permit conjugation.
In certain embodiments, the PARPi-fl has a molecular weight no greater than about 1000 Da (e.g., no greater than 900 Da, e.g., no greater than 800 Da).
In certain embodiments, the PARPi-fl has at least a moderate lipophilicity (e.g., to permit penetration into a cell nucleus).
In certain embodiments, the fl moiety having a molecular weight no greater than about 500 Da, e.g., no greater than about 400 Da, e.g., no greater than about 300 Da.
In certain embodiments, the kit further comprises 18F-PARPi.
In another aspect, the invention is directed to a composition comprising PARPi-fl, wherein fl comprises a fluorophore, for use in (a) a method of in vivo diagnosis of cancer in a subject with oral squamous cell carcinoma (OSCC) or (b) a method of assessing a cancer therapy in a subject, wherein the method comprises: delivering the composition onto and/or into tissue in an oral cavity of the subject; optionally, flushing the tissue in the oral cavity of the subject to reduce or remove unbound components of the composition while leaving bound components of the composition; and optionally, detecting fluorescence from the fluorophore after the administering and the flushing steps, thereby identifying potential OSCC.
In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.
In this application, the use of “or” means “and/or” unless stated otherwise. As used in this application, the term “comprise” and variations of the term, such as “comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps. As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Headers are provided for convenience only and are not intended to limit the content or applicability of the material thereunder.
“Administration”: The term “administration” refers to introducing a substance into a subject. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, intramuscular, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is oral. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous.
“Biocompatible”: The term “biocompatible”, as used herein is intended to describe materials that do not elicit a substantial detrimental response in vivo. In certain embodiments, the materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce inflammation or other such adverse effects. In certain embodiments, materials are biodegradable.
“Biodegradable”: As used herein, “biodegradable” materials are those that, when introduced into cells, are broken down by cellular machinery (e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo. In some embodiments, biodegradable materials are enzymatically broken down. Alternatively or additionally, in some embodiments, biodegradable materials are broken down by hydrolysis. In some embodiments, biodegradable polymeric materials break down into their component polymers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymeric materials) includes hydrolysis of ester bonds. In some embodiments, breakdown of materials (including, for example, biodegradable polymeric materials) includes cleavage of urethane linkages.
“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. In some embodiments, the composition described herein is a carrier.
“Non-invasive”: As used herein, the term “non-invasive” refers to methods that are non-surgical, e.g. not penetrating the body, as by incision or injection, or not invading tissue.
In some embodiments, topical administration of a composition to a surface of a tissue is understand as a non-invasive technique.
“Subject”: As used herein, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are be mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.
“Therapeutic agent”: As used herein, the phrase “therapeutic agent” refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.
“Treatment”: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
Drawings are presented herein for illustration purposes, not for limitation.
The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conduction with the accompanying drawings, in which:
bar: 50 μm.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.
The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.
Described herein are methods of using radiolabeled or fluorescent poly(ADP-ribose)polymerase 1 (PARP1) imaging probes with high selectivity and specificity to detect the cancer biomarker PARP1 in the oral cavity via topical application, e.g. in a dentist office setup using a macroscopic fluorescence scanning imaging device. After topical application of PARPi-fl to the oral cavity, the imaging probe accumulates only in areas of elevated PARP1 expression. Employment of a microscopic device, such as a fluorescence endoscope or a handheld confocal microscope can improve identification of tumor margins and residual tumor tissue during tumor removal surgery.
Earlier and more accurate detection of oral squamous cell carcinoma (OSCC) is essential to improve the prognosis of patients and to reduce the morbidity of surgical therapy. It was demonstrated herein that PARP1 is a target for optical imaging of OSCC with the fluorescent dye PARPi-fl. In patient-derived OSCC specimens, PARP1 expression was increased 7.8±2.6-fold when compared to normal tissue. Intravenous injection of PARPi-fl allowed for high contrast in vivo imaging of human OSCC models in mice with a surgical fluorescence stereoscope and high-resolution imaging systems. The emitted signal was specific for PARP1 expression, demonstrating that PARPi-fl can be used as a topical imaging agent, spatially resolving the orthotopic tongue tumors in vivo. The results suggest that PARP1 imaging with PARPi-fl can enhance the detection of oral cancer, serve as a screening tool and help to guide surgical resections.
Vital signs can be monitored before PARPi-fl administration and after completion of imaging. The oral mucosa can be checked for local irritation, after completion of imaging and up to about 3 days later. Two blood samples can be obtained prior to PARPi-fl imaging and up to about 3 days after PARPi-fl administration.
Radiolabeled and optically active 18F-PARPi and PARPi-fl were described in “Compositions and methods for in vivo imaging” by Keliher et al., in International Publication Number WO/2012/074840 A2, which is hereby incorporated by reference (See Appendix A). These imaging probes possess favorable pharmacokinetic properties to detect and interrogate tumor growth and treatment success. Moreover, they are compatible with current widely used imaging technologies in laboratory and diagnostic medicine— PET and fluorescence imaging.
As described herein, PARPi-fl and 18F-PARPi are pharmacologically identical agents that accumulate quickly and selectively in cancer cells that overexpress PARP1, and can therefore serve as a screening tool to noninvasively delineate the presence and extent of neoplastic growth in the oral cavity. The functions of PARPi-fl and 18F-PARPi are complimentary. Although 18F-PARPi lacks the high resolution and sensitivity of optical imaging, it enables detection of deep seated lesions such as lymph node and distant metastases. Complimentary to 18F-PARPi, PARPi-fl highly permeates the cells, providing more accurate (sensitive and specific) detection of oral cancer based on the high PARP1 expression of oral squamous cell carcinoma cells. Furthermore, as disclosed herein, the use of PARPi-fl enables cellular-resolution point-of-care imaging and does not require expensive infrastructure and specialized personnel to accurately and non-invasively screen for lesions with high spatial accuracy. Used together, PARPi-fl and 18F-PARPi are highly sensitive and specific diagnostic tools that can be used to detect oral squamous cell cancers. Thus, the two orthogonal imaging modalities (e.g. PET and optical imaging) enable screening (e.g., via optical imaging) and staging (e.g., via PET imaging) of this disease.
The PARP1 imaging agents disclosed herein can be used as diagnostic markers for the early detection of oral cancer in the oral cavity (e.g., of a human). The fluorescent PARPi-fl can be used as an optical imaging agent for the screening and diagnosis of squamous cell carcinoma of the oral mucosa (e.g., of a human). Moreover, the 18F-PARPi can be used as a quantitative PET imaging agent to assist non-invasive diagnoses.
Various mouse models, described herein, have been used to quantify PARP1 expression in OSCC. For example, xenografts and orthotopic mouse models of oral cancer, induced by the injection of human OSCC cancer cells into the tongue bed, have been studied as described herein. Moreover, a model can rely on chemically induced oral cancer (e.g. addition of 4-nitroquinoline 1-oxide to drinking water) or a blinded study can be performed to determine the sensitivity and specificity of PARPi-fl for oral cancer tissue.
PARPi-fl and 18F-PARPi can be used in human cell line models of OSCC including subcutaneous xenografts, orthotopic xenografts, and chemically induced oral cancer. The pharmacokinetics of PARPi-fl and 18F-PARPi are also disclosed herein. In some embodiments, agarose phantoms, xenografts and orthotopic models of OSCC for epifluorescence imaging, autoradiography, and PET imaging can be used to determine the correlation curves and quantitative analysis for these imaging techniques.
As shown in in
An in vivo administered dose of 18F-PARPi and/or PARPi-fl can confirm malignant lesions based on the high expression of PARP1 in highly proliferative tissue. As described herein, PARPi-fl is a valuable tool for the detection of OSCCs in cancer. Moreover, this agent can be used to screen for developing premalignant lesions. Furthermore, its sister imaging agent, 18F-PARPi, can be a powerful tool to quantify the extent of malignant growth below the tissue surface, at local or even distant sites. A therapeutic PARP1 inhibitor can also be administered with a PARP1 imaging agent. The therapeutic PARP1 inhibitor can be AG014699 (rucaparib), ABT888 (veliparib), BSI201 (iniparib), BSI101, DR2313, FR 247304, GPI15427, GPI16539, M 4827 NU1025 NU1064, NU1085, P1)128763, PARP Inhibitor H (INH2BP), PARP Inhibitor m (DPQ), PARP Inhibitor VIII (PJ34), PARP Inhibitor IX (EB-47), and TIQ-A, as described by Keliher et al.
Therefore, use of these bimodal imaging probes facilitate diagnosis of this cancer which enables adoption of the technology by healthcare professionals.
Although the genetic and molecular basis of cancer and its diagnosis via non-invasive imaging has advanced significantly over the last several decades, early diagnosis and noninvasive detection of OSCC remain limited. It appears that no models of human cancer in the oral cavity have been heretofore validated.
It is described herein that PARP1 is a specific and selective early biomarker for the detection of OSCC. Primary human biospecimen of oral cancer were obtained and analyzed using standard clinical pathology and grouped into healthy, premalignant and malignant tissues. Part of the biopsied tissue were used to determine the PARP1 expression. This provided corroboration of to what degree PARP1 expression is elevated in human oral cancer.
PARP1 expression is highly upregulated in mouse models of oral cancer as shown in
Similar to most therapeutic small molecules, 18F-PARPi and PARPi-fl bind to the NAD+ binding site of PARP1. The imaging agents can therefore be used as companion imaging agents for PARP1 inhibitors that are binding to the same location (e.g. ABT-888, Abbott; AG014699, Pfizer; AZD2281, Astra-Zeneca; BMN-673, Biomarin; MK-4827, Merck). PARP1 imaging allows physicians to stratify patients in their appropriate treatment groups, enabling clinical decision making processes based on PARP1 levels.
In combination, these bimodal imaging agents can be used to leverage the unique properties and selective accumulation of these small molecules in proliferative growths. Therefore, when in clinical use, the optical component of PARPi-fl can be used to screen for the presence of oral cancer (which in more than 90% of all cases occurs direct at the tissue surface. Once suspected or confirmed, the PET component of 18F-PARPi can be used to quantify the exact tumor burden and determine whether the cancer is local or has metastasized.
The fluorescence signal for PARPi-fl stems from the fluorophore. For example, in the case where the fluorophore is BODIPY®-FL, the dye BODIPY-FL emits fluorescent light of wavelength of 525 nm when excited at 488 nm. The PARPi-fl composition has a similar binding affinity to PARP1 as the olaparib ((IC50 for inhibition of PARP1 enzymatic activity 12.5 nM vs. 6.0 nM for olaparib).
In addition to PET imaging, confocal microscopy of the oral cavity can be used to image the bimodal agent PARPi-fl for fluorescence in vivo. Although PET is a highly sensitive imaging modality for whole-body screening, it requires large infrastructure and lacks the ability to image suspicious lesions noninvasively at the sub-cellular level. Moreover, while wide-field imaging is beneficial for rapid surveillance of an entire oral cavity, it generates a large number of “false-positive” results obtained by such wide-field approaches.
Thus, a miniature, portable confocal microscope that rapidly obtains images of glandular, cellular, and nuclear detail for diagnosing suspicious tissues in vivo, and guides the acquisition of excisional biopsies can be used for screening and detecting tumors in the oral cavity. This optical sectioning technology can both improve the early detection of oral cancers, as well as significantly reduce the time, cost, and patient discomfort associated with the acquisition of large numbers of unnecessary biopsies.
Expression levels of PARP1 are in the micromolar range and thus higher than of many other proteins upregulated in cancer.
Referring back to
Acid catalyzed 18F/19F exchange allows formation of 18F-PARPi. In the presence of a strong Lewis Acid, a cold 19F atom attached to BF2 group of PARPi-fl can be replaced with a PET active 18F. 18F-PARPi was synthesized using an automated synthesis module as shown in
Similar to the mouse models of glioblastoma shown in
Mice were inoculated orthotopically with a fluorescent oral cancer cell line (tdTomato-FaDu and tdTomato-Ca127). PARPi-fl imaging agent uptake in the tumor was observed with an IVIS preclinical imaging system (Perkin Elmer, Waltham, Mass.) and compared to the expression of the fluorescent cell line. A correlation of PARPi-fl and tumor growth can be assumed if the Pearson's correlation coefficient between both fluorescent channels is greater than 0.95.
Blind Detection in Chemically Induced Models of OSCC with PARPi-fl
A chemically induced model of OSCC was used to determine the sensitivity of PARPi-fl for detecting the presence of malignant lesions. For this model, the presence of disease was determined histologically in three categories (cancer, pre-cancer, normal). One hundred mice were used for this purpose. Three separate readers who were blinded to visible light images. Histology results then read the set of fluorescence imaging data in randomized order and determined whether or not, based on a 5-point scale, cancerous tissue was present.
The blood half-life, serum stability, and metabolic stability of 18F-PARPi was determined. Blood half-lives were determined after the injection of ˜15 μCi of the 18F-labeled species into C57BL/6J (B6) mice and blood (5-10 μL) drawn at different time points via saphenous vein bleeds.
The uptake and clearance rates of 18F-PARPi in xenograft, orthotopic and chemically induced oral cancer was measured. For xenografts, cancer cells (1-5×106 cells in 1:1 PBS:BD Matrigel for mouse xenografts and 5×104 cells for orthotopic models) were injected and the tumors grew for 5-10 days for xenograft models and 12-20 days for orthotopic cancers. For chemically induced OSCC, tumors were induced through the addition of 4-nitroquinoline 1-oxide to drinking water. Comparison of the % ID/g in tumors and healthy oral tissues as well as excretory organs, bone, urine and feces at different injected amounts of the tracer (300 Xi-500 Xi, 0.5 μg-100 μg injected material) were measured at different time points.
In order to assess specificity of the tracer uptake, blocking experiments with both cold PARPi-fl as well as the inhibitor olaparib were designed. Comparison and quantification of PARPi-fl and 18F-PARPi biodistribution were performed. Agarose phantoms were produced and 18F-PARPi (at 5 μCi-5000 Xi) and PARPi-fl (0.5 nmol-500 nmol) were imaged to create calibration curves for the bimodal imaging probe system. Imaging of the signal strength using an epifluorescence imaging system, autoradiography, and PET was performed. The resulting calibration curves allowed quantification of the uptake and emission of the imaging agents, and to determine the amount of inhibitor in a given volume. A range of 18F-PARPi activities (300 mCi-500 mCi) and PARPi-fl concentrations (25 nmol-75 nmol) were injected in mice. The animals were imaged at various time points using all imaging modalities.
PARP1 expression in human OSCC cell lines in a subcutaneous xenograft mouse model using Immunofluorescence (IF) PARP1 staining is depicted in
The specific uptake of PARPi-fl into these cell lines can be shown in vitro, and can also be confirmed in subcutaneous xenograft models using epifluorescence imaging. Disclosed herein, the accumulation of PARPi-fl in tumor cells but not normal tissue led to high tumor to background ratios (tumor to muscle ratios; FaDu: 4.6±1.4, Ca127: 2.9±1.0). In concordance with a higher PARP1 expression, PARPi-fl accumulation was higher in FaDu than in Ca127. In support of the disclosed intraoperative approach, PARPi-fl accumulation was shown in whole excised xenografts. PARPi-fl accumulation can also be detected using a fluorescence endoscope and a custom built dual-axis confocal microscope, in the form of a large or handheld or portable device.
Next, the imaging approach was tested in an orthotopic tongue tumor model using FaDu cells. This approached confirmed that sufficiently high PARPi-fl accumulation compared to the surrounding healthy tissue was able to be detected in tongue tumors. As shown in
The above examples have shown in models of cancer that PARPi-fl, a fluorescent PARP1-targeted small molecule, specifically binds to PARP1 with a similar affinity to olaparib (Lynparza, Astra-Zeneca), an FDA-approved PARP1 inhibitor. These studies were performed in cell culture and with ex vivo imaging of excised tumors.
The expression of PARP1 in human OSCC was first determined using biospecimens from OSCC patients and identified mouse models of OSCC that reflect the human disease, including the expression levels of PARP1. PARPi-fl in these mouse models was tested and confirmed to be clinically relevant, non-invasive imaging systems that are capable of visualizing OSCC with high contrast after both intravenous and topical administration of PARPi FL. Without wishing to be bound to any theory, this suggested that PARPi-fl can be used to answer diagnostically relevant questions in the clinic.
To demonstrate that high expression of PARP1 exists in human oral cancer specimen, human tongue tumor biopsies for PARP1 were used for Immunohistochemistry (n=10, 3 specimens each in tumor stage T2, T3 and T4). It was found that there is a distinction between PARP1 expression in malignant tissue compared to adjacent normal tissue areas. Quantification of the PARP1 positive area showed that in the mean of all human samples, 18.0±4.9% of the tumor area is PARP1 positive, whereas in healthy tongue tissue only 2.9±1.9% of the tissue area shows PARP1 expression (p<0.0001, students t-test). Looking at individual samples, the PARP1 positive area is markedly higher in each sample as shown in
PARP1 Expression in Human Oral Cancer Biospecimens
To determine the relevance of PARP1 as a biomarker for OSCC, PARP1 expression patterns in human oral cancer tissues, along with PARP1 expression in adjacent healthy tissues in 12 human tongue tumor specimens were obtained from the Department of Pathology at Memorial Sloan Kettering Cancer Center (MSK), which were histopathologically staged using H&E stained biopsy tissue following the standard tumor, node, metastasis (TNM) classification.
The tissues included three specimens per tumor stage: premalignant, T2, T3, and T4 (Table 1). The premalignant tissues were classified as moderate/severe dysplasia and squamous cell carcinoma in situ. The three specimens per tumor stage except for one were obtained from the edges of the tumors and featured both tumor tissue as well as healthy surrounding tissue (
Differences in PARP1 expression was also observed when comparing different tumor stages, albeit these differences in PARP1 expression are based on a small sample size (n=3 per tumor stage) (
The performance of PARP1 as a classifier for tumor and normal tissue was evaluated using a receiver operating characteristic (ROC) curve (
Expression of PARP1 was found to be similar in two xenograft models of human OSCC (
Ex Vivo PARP1 Imaging with PARPi-fl in Subcutaneous OSCC Xenografts
Next, it was determined whether FaDu and Ca127 tumors accumulated PARPi-fl after intravenous injection, and if tumor uptake was due to binding to PARP1. PARPi-fl is a targeted imaging agent that fluoresces in the visible range (
The fluorescence signal was quantitatively evaluated by tissue-to-thigh-muscle ratios. This ratio was 4.6±1.4 for FaDu tumors and 2.9±1.0 for Ca127 tumors (
Following IV injection in mice, PARPi-fl was cleared rapidly from the circulation with an a half-life of 1.2 min and a β half-life of 88 min. PARPi-fl was rapidly taken up by cancer cells in tumor xenografts and reaches the nucleus within minutes (
PARP1 antibody staining was highly co-localized with PARPi-fl fluorescence (Rcoloc.=0.986, R2=0.973; 95% confidence interval 0.98 to 0.989;
PARPi-fl uptake was also imaged in vivo in an orthotopic tongue tumor model of OSCC (FaDu cells) using the same parameters as for subcutaneous tumor imaging ex vivo (intravenous injection of 75 nmol PARPi-fl/animal, imaging 90 minutes post-injection). Here, epifluorescence imaging showed a strong PARPi-fl accumulation in parts of the tongue that were visibly affected by OSCC, whereas no signal accumulation was observed in tongues without tumors after PARPi-fl or vehicle injection (
To show that PARPi-fl is suitable for imaging of tumors at cellular resolution, freshly excised FaDu tumor tissue were imaged 90 minutes after injection of PARPi-fl using a custom dual-axis confocal microscope at a range of depths (
Oral Cancer Imaging after Topical Application of PARPi-fl
Topical application of a PARPi-fl formulation (30% PEG300/PBS) with subsequent fluorescence screening of the oral cavity for OSCC detection can improve the current standard of care, particularly in low resource settings. For example,
In a preclinical model of tongue OSCC, it was investigated if PARPi-fl tumor contrast after topical application was comparable to intravenous injection. Macroscopic evaluation using epifluorescence imaging confirmed that PARPi-fl colocalized to areas of the tongue where there was the orthotopic tumor, as confirmed by tumor cells expressing the fluorescent protein tdTomato (
These results indicate that PARP1 protein expression is markedly increased in OSCC when compared to normal tissues of the oral cavity. Moreover, it was demonstrated that the small molecular imaging agent PARPi-fl can be used to delineate OSCC in living mice. PARPi-fl is efficiently retained in oral cancer tissue, yielding a strong imaging signal, paired with high contrast to surrounding normal tissue. This enabled high-resolution in vivo imaging of orthotopic OSCC with clinically translatable instruments. Using these devices, PARP1 expression was imaged from the macroscopic to the subcellular level. It was also shown that PARPi-fl, due to its high tissue permeability (4.7±2.5 μm/s), efficiently penetrates into tumor tissue after topical application, and selectively accumulates in OSCC cells close to the tissue surface, while being washed out from non-target tissues and compartments within minutes.
PARP1 expression was elevated throughout the patient-derived OSCC samples. PARP1 expression per nucleus was fairly uniform. However, the density of PARP1-positive tumor cells varies in different areas. Specifically, PARP1 expression levels were higher at the invasive margins of the tumors than in the center. The impact of tumor cell density is also apparent in
PARP1 is expressed in a large number of cancers. Other members of the PARP family, such as PARP2, which is also inhibited by olaparib, is less abundant and its expression was found not to be upregulated in a number of primary cancers. Although, no data on PARP2 expression in oral cancer are currently available, this is pointing towards a less important role of PARP2 in tumorigenesis and a low suitability as cancer imaging agent compared to PARP1. Without wishing to be bound to any theory, PARP1 overexpression may be due to the increased DNA damage occurring in genetically unstable cancer cells, rather than the activation of specific oncogenic pathways. Furthermore, the density of nuclei is typically higher in malignant tumors than in most normal tissues. The PARPi-fl in vivo imaging signal therefore reflects both the higher expression levels of PARP1 per nucleus as well as the higher nuclear density in malignant tumors. Thus, PARPi-fl can be used to image a large variety of tumors during screening or surgery. OSCC is an obvious candidate for the initial evaluation of PARPi-fl imaging because of the clinical needs for better detection and delineation of OSCC, as well as its easy accessibility for fluorescence imaging.
In the field of optical fluorescence imaging, a large number of probes absorb and emit near-infrared light. In this wavelength range (650 nm-900 nm), photons are less scattered and absorbed by tissues, which allows for better tissue penetration. In addition, there is less background autofluorescence from tissues with near-infrared excitation, as compared to visible excitation. The BODIPY® FL fluorophore used to synthesize certain experimental embodiments of PARPi-fl operates in the visible range of light (400 nm-700 nm), but it has the added advantage of an exceptionally low molecular weight and it does not ionize under physiological conditions. Other fluorophores may also have these advantages. These properties allow for efficient in vivo extravasation, combined with fast cell permeation and intranuclear accumulation. Larger fluorophores and charged molecules result in significantly reduced cell permeability, together with low contrast ratios. The in vivo imaging results presented here confirm that the fluorescence from the fluorophore allows for high-contrast imaging of superficial tumors in the tongue despite the known limitations of green fluorescent dyes. Moreover, fluorescence imaging systems that operate with a green fluorescence channel have entered clinical practice, e.g., probe based confocal laser endomicroscopy (pCLE), which is FDA-approved for imaging the entire gastrointestinal tract, including the oral cavity. The utility of pCLE imaging for better differentiation of nondysplastic, precancerous, and cancerous lesions of the head and neck in patients has already been shown using fluorescein, a nonspecific green fluorescent dye, which absorbs and emits very closely to PARPi-fl (Excitation/Emission max.; fluorescein: 490/525 nm; PARPi-fl: 507/525 nm). PARPi-fl accumulation in OSCC was imaged with high contrast using a clinically approved pCLE system.
Using a PARPi-fl assisted oral cancer screening procedure, the application method of PARPi-fl can be switched from intravenous to topical application, reducing complexity, and increasing the agent's breadth and versatility in the clinic. Topical application further reduces cost and potential side effects, and streamlines the imaging protocol. Optical fluorescence imaging equipment is lower priced and has a higher grade of mobility compared to other molecular imaging modalities, for example PET or MM.
PARPi-fl was shown to penetrate up to 300 μm into tissue, which is sufficient for detection of OSCC, a disease that typically originates within the outermost cell layers of the oral cavity. In conclusion, the results described herein indicate that PARPi-fl imaging of OSCC is very promising for a variety of applications, including cancer screening, surgical guidance during tumor removal, and delineation of tumor margins by pCLE. Hence, PARP1 imaging can result in earlier detection of oral cancer and reduce the morbidity of radical surgery that plagues patients suffering from OSCC.
The toxicity of several BODIPY-FL labeled molecular imaging agents has been evaluated in cell culture studies. Toxic effects were only observed after prolonged exposure at concentrations of 10 μM or more. The toxicity of PARPi-fl was compared with Olapirib in two glioblastoma cell lines (e.g., U87 and U251). The IC50s in an MTT assay for olaparib and PARPi-fl were 28 μM and 24 μM in U87 cells, and 8.0 and 5.5 in U251 cells, indicating that in these two cell lines PARPi-fl was not more toxic than olaparib.
Local administration of 20 μl of a 400 μM solution of PARPi-fl was tested on the oral mucosa of mice. This dose caused no local irritation, and no changes in clinical chemical and hematologic parameters (Tables 2A and 2B).
Tables 2A (Clinical chemistry) and 2B (Hematology) shows toxicity of PARPi-fl after local administration on the oral mucosa of mice. Cohorts of mice (6-8 weeks old female athymic mice) were administered a solution of PARPi-fl as a topical application (29 nmol PARPi-fl in 50 μL), and incubated for 10 min, before excess agent was washed off. Mice received blood draws after 24 h and 48 h post administration, and were then sacrificed to receive a full necropsy.
There is no clinical experience with PARPi-fl or BODIPY-FL. However, there are data on the FDA approved PARP1 inhibitor olaparib which is the PARP1 binding motif of PARPi-fl. These are briefly summarized in the following paragraphs.
The approved dose of olaparib for treatment of ovarian cancer is 400 mg orally twice daily. Treatment is typically given continuously over several months. Following oral administration olaparib is rapidly absorbed with peak plasma concentrations between 1-3 hours after dosing. The apparent volume of distribution is more than 150 L, indicating intracellular accumulation. olaparib is metabolized via CYP34A and the metabolites are excreted via urine and bile. The terminal half-life is 11.9 hours after administration of a 400 mg dose. More than 86% of 14C-labeled olaparib was excreted within 7 days.
At the approved dose and dose schedule olaparib is well tolerated. Reported side effects in patients with advanced ovarian cancer being treated with olaparib include anemia, abdominal pain, decreased appetite, nausea, vomiting, diarrhea, dyspepsia and pharyngitis.
The OSCC cell lines FaDu (hypopharyngeal SCC; ATCC, Manassas, Va.) and Ca127 (tongue SCC; ATCC, Manassas, Va.) were grown in a monolayer culture at 37° C. in a 5% CO2 humidified atmosphere. FaDu cells were maintained in MEM medium and Ca127 cells in D-MEM medium, both containing 10% (v/v) FBS and 1% PenStrep.
Female athymic nude mice (NCr-Foxn1nu, Taconic, Hudson, N.Y.) were housed under standard conditions with water and food ad libitum. Throughout all procedures, animals were anesthetized with 2% isoflurane. To implement subcutaneous human OSCC tumors, 2×106 FaDu or Ca127 cells were dispensed in 100 μl of a 1/1 mixture of medium/Matrigel™ (BD Biosciences, Bedford, Mass.) and were injected into the lower back of the animals. Experiments were conducted when tumors reached 100-150 mm3 volume. For an orthotopic OSCC model, 5×105 FaDu or FaDutdTomato (FaDu stably transfected with tdTomato fluorescent protein; Creative Biogene, Shirley, N.Y.) cells in 20 μl PBS were injected directly into the tongue and the mice were observed daily for tumor growth and weight loss. Imaging was conducted usually after 3-4 weeks. All animal experiments were performed in accordance with institutional guidelines and approved by the IACUC of MSK, and followed NIH guidelines for animal welfare.
Quantification of PARP1 expression was carried out using human tongue tumor specimens (n=12), obtained from the Department of Pathology of MSK. The use of tissues was approved by the Institutional Review Board (IRB) at MSK and informed consent was obtained from all subjects.
PARP1 antigen in human oral cancer tissue, as well as FaDu and Ca127 xenografts and mouse tissues was detected using immunohistochemical (IHC) and immunofluorescence (IF) staining techniques, which were performed at the Molecular Cytology Core Facility of MSK using the Discovery XT processor (Ventana Medical Systems, Tucson, Ariz.). The anti-PARP1 rabbit polyclonal antibody (sc-7150, Santa Cruz Biotechnology, Santa Cruz, Calif.) specifically bound both human and mouse PARP1 (0.2 μg/ml). Paraffin-embedded formalin fixed 3 μm sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer (both Ventana Medical Systems, Tucson, Ariz.), and sections were blocked for 30 minutes with Background Buster solution (Innovex, Richmond, Calif.). Anti-PARP1 antibody was incubated for 5 hours, followed by 1 hour of incubation with biotinylated goat anti-rabbit IgG (PK6106, Vector Labs, Burlingame, Calif.) at a 1:200 dilution. For IHC detection, a DAB detection kit (Ventana Medical Systems, Tucson, Ariz.) was used according to the manufacturer's instructions, sections were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific, Pittsburgh, Pa.). IF detection was performed with Streptavidin-HRP D (from DABMap Kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 594 (T20935, Invitrogen, Carlsbad, Calif.) prepared according to the manufacturer's instructions. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes and coverslipped with Mowiol® mounting medium (Sigma-Aldrich, St. Louis, Mo.). Incubating with a rabbit IgG instead of the primary antibody controlled for non-specific binding of the secondary antibody. For morphological evaluation of tissue characteristics, H&E staining was performed on adjacent sections.
For PARP1 protein quantification, stained tumor sections were digitalized using a MIRAX Slide Scanner (3DHISTECH, Budapest, Hungary). On at least 10 fields of view per section, PARP1 presence was quantified using MetaMorph® Software (Molecular Devices, Sunnyvale, Calif.). In IHC stained tissues, a thresholding was performed on brown (PARP1) and blue (tissue) areas and the relative PARP1-positive area was calculated by dividing the brown area by the total tissue area. For IF, the PARP1-positive area was determined by thresholding the red fluorescent area and dividing it by the whole tissue area, which was determined based on autofluorescence in the green channel. PARP1 intensity was also determined by measuring the red fluorescence intensity in all nuclei, which were thresholded using DAPI staining. The measured fluorescence intensities were averaged over all nuclei in each field of view, with intensity values ranging from 0-255.
Synthesis of the optical imaging agent PARPi-fl was carried out as described herein. The green fluorescent dye BODIPY-FL NETS-ester (Invitrogen, Carlsbad, Calif.) was conjugated to 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one and purification by preparative HPLC (Waters' XTerra C-18 5 μm column, 7 ml/min, 5% to 95% of acetonitrile in 15 min) afforded PARPi-fl in 70-79% yield as a red solid. Analytical HPLC analysis (Waters' Atlantis® T3 C18 5 μm 4.6×250 mm column) showed high purity (>97%) of the imaging agent. The identity of PARPi-fl was confirmed using ESI-MS (MS(+) m/z=663.4 [M+Na]+). For imaging studies, PBS (117 μl) was slowly added to an aliquot of PARPi-fl (50 μg, 75 nmol) in 50 μl of poly(ethylene glycol) (PEG300, Sigma-Aldrich, St. Louis, Mo.) to obtain a final injection volume of 167 μl.
For evaluation of the uptake of PARPi-fl in subcutaneous OSCC xenografts, animals carrying either FaDu or Ca127 tumors were intravenously injected with PARPi-fl (75 nmol/167 μl PBS with 30% PEG300 (Sigma-Aldrich, St. Louis, Mo.)) (n≥6/group). To assess the specificity of PARPi-fl accumulation in one group of animals, a 50-fold excess (3.75 μmol/100 μL PBS with 30% PEG300) of olaparib (LC Laboratories, Woburn, Mass.) was injected 30 minutes prior to the PARPi-fl injection, blocking the specific binding sites in FaDu tumors (n=4). Animals were sacrificed 90 minutes post-injection and tumors, tongues, trachea, and muscle were excised and imaged using epifluorescence imaging (IVIS Spectrum, PerkinElmer, Waltham, Mass.). For detection of the fluorescent PARPi-fl emission, a predefined GFP Filterset (excitation: 465/30 nm, emission: 520-580 nm) was used and subsequently removed autofluorescence through spectral unmixing. Semiquantitative analysis of the PARPi-fl signal was conducted by measuring the average radiant efficiency in regions of interest (ROIs) that were placed on all organs under white light guidance. This measure carries the unit [p/s/cm2/sr]/[μW/cm2] and is defined as the number of photons per second leaving a square centimeter of tissue and radiating into a solid angle of one steradian (sr). Resulting numbers are normalized for the integration time, binning, f/stop, field of view, illumination intensity, and the ROI area, making measurements comparable among each other. Freshly excised whole tumors were also microscopically imaged directly after epifluorescence imaging; tissues were placed on a cover slip with a freshly cut surface facing the cover slip and images were taken on an inverted laser scanning confocal microscope using 488 nm laser excitation (LSM 5-Live, Zeiss, Jena, Germany).
To determine the specificity of the accumulation of PARPi-fl within tumor tissue, the inter- and intracellular co-localization of the targeted fluorescent probe with PARP1 antigen was determined in histological sections. FaDu xenografts and control tissues (tongue, muscle) were snap-frozen 90 minutes after intravenous injection of PARPi-fl (75 nmol/167 μl 30% PEG300 in PBS). Next, 10 μm cryosections were fixed in 4% paraformaldehyde for 8 minutes, followed by blocking with 3% (v/v) goat serum (Sigma-Aldrich, St. Louis, Mo.) in PBS. Antibodies were diluted in 1% (w/v) BSA and 0.3% (v/v) Triton X-100 in PBS. Anti-PARP1 primary antibody (sc-7150, Santa Cruz Biotechnology, Santa Cruz, Calif.) was incubated overnight at 4° C. (1 μg/ml), followed by three 10-minute washes with PBS and incubation with secondary AlexaFluor® 680 goat anti-rabbit antibody (A21076, Molecular Probes, Eugene, Oreg.) for 1 hour at 4° C. (2 μg/ml). After another 5-minute PBS wash, sections were mounted with Mowiol® (Sigma-Aldrich, St. Louis, Mo.) containing Hoechst 33342 DNA Stain (Sigma-Aldrich, St. Louis, Mo.). Fluorescence images were captured using a Leica (Buffalo Grove, Ill.) SP8-inverse confocal microscope equipped with a 405 nm laser for detection of cell nuclei, a 488 nm laser for detection of in vivo applied PARPi-fl, and a 670 nm laser for detection of PARP1 antibody stain, each paired with suitable emission filters. Incubating sections with either a nonspecific rabbit IgG or PBS instead of primary antibody confirmed binding specificity. Bleed-through of signals into other channels was excluded by imaging sections that were either not injected with PARPi-fl in vivo (no signal should be seen in the 488 nm channel) or not stained with PARP1 (no signal should be seen in the 670 nm channel). Correlation analysis between PARPi-fl and PARP1 signal intensity was performed using MetaMorph® Software (Molecular Devices, Sunnyvale, Calif.).
For orthotopic FaDu tongue tumors, epifluorescence imaging was conducted using the same procedure as described above, but animals were alive when imaged 90 minutes post-injection. All animals were anesthetized with 2% isoflurane in medical air. The tongues of all animals were exposed by opening their mouths and moving the tongue past the front teeth into the field of view of the IVIS. Animals were divided into three groups: tumor-bearing animals that were injected with PARPi-fl (75 nmol/167 μl PBS with 30% PEG300), healthy animals that were injected with PARPi-fl, and healthy animals that were injected with vehicle (167 μl PBS with 30% PEG300) (n=3/group). Afterwards, animals were sacrificed and tongues, trachea, and thigh muscle were imaged ex vivo. Using the same experimental setup, imaging with a fluorescence stereoscope was conducted to show that the PARPi-fl signal was also be detected under real-time imaging conditions, as would be the case in the clinical setting. Here, the tongues of anaesthetized animals were imaged using 500/20 nm excitation and 535/30 emission filters and a fixed exposure time of 500 ms (SteREO Lumar.V12, Zeiss, Jena, Germany). Imaging was performed 90 minutes after intravenous injection of PARPi-fl (75 nmol/167 μl PBS with 30% PEG300).
To show the feasibility of intravital tumor imaging at cellular resolution, excised subcutaneous FaDu xenografts, tongue, and muscle were imaged 90 minutes after PARPi-fl (75 nmol/167 μl PBS with 30% PEG300) or vehicle injection using a custom-built dual-axis confocal microscope. Illumination settings were optimal for BODIPY-FL imaging and settings (laser intensity and detector gains) were fixed for all tissues to ensure comparability (illumination intensity: 1.95-2.1 mW and photomultiplier gain setting: 0.656 V).
FaDu xenografts were imaged with a fluorescence endoscope that is available for both clinical and preclinical imaging (Cellvizio, Mauna Kea Technologies, Paris, France). It provides cellular to subcellular resolution and has a flexible confocal microprobe that enables versatile imaging. Here, after receiving a 90-minute post-injection of 150 nmol PARPi-fl (in 167 μl PBS with 30% PEG300) or vehicle (167 μl PBS with 30% PEG300), animals were sacrificed and skin was removed from subcutaneous FaDu tumors and thigh muscle (n=4 PARPi-fl, n=3 vehicle). The microprobe was slowly moved over the tumor, tongue, or muscle, while a real-time video was recorded using a 488 nm excitation beam. The videos were converted to grayscale and the intensity was measured in 10 frames per video using ImageJ 1.49e Software. Topical application of PARPi-fl
For topical application of PARPi-fl, mice with or without orthotopic tongue tumors (FaDutdTomato) were anaesthetized using ketamine (0.1 mg/g body weight) and tongues were exposed using forceps. For topical application, the tongues were dipped into a well of a 96-well plate filled with the respective incubation solution. The sequence of incubation was first 20 seconds in 1% acetic acid second 20 seconds PBS third 1 minute 5 μM PARPi-fl (30% PEG300/PBS) fourth 1 minute 1% acetic acid and fifth 10 seconds PBS. This was followed by cleaning of the tongue with an alcohol pad to remove residual unbound compound. The animals were imaged in the IVIS Spectrum before and after PARPi-fl application using the appropriate filter sets for detection of PARPi-fl and the tdTomato fluorescent protein. Spectral unmixing was used to separate the signals for tdTomato, PARPi-fl and autofluorescence. The tdTomato fluorescent protein allows in vivo localization of the tumor. For comparability, all images were scaled to the same maximum radiant efficiency. Imaging was repeated with sections of the excised tongues after cryofixation. Sections were fixated in 4% PFA, counterstained with Hoechst and imaged using a confocal microscope to localize PARPi-fl in the tissue. Adjacent sections were H&E stained for morphological evaluation.
Statistical analysis of preclinical data was performed using GraphPad Prism 6 and R 3.1 (www.r-project.org). Unless otherwise stated, data points represent mean values, and error bars represent standard deviations of biological replicates. P values were calculated using a Student's unpaired t-test, corrected for multiple comparisons by the Holm-Sidak method with an alpha of 0.05 as the cutoff for significance. For the clinical specimen, the distribution of the percent PARP1-positive area was separately estimated for normal and malignant tissues using kernel density estimation. The ability to use PARP1 expression to distinguish malignant tissues from adjacent normal tissue was characterized by a receiver operating characteristic (ROC) curve. The probability of a given tissue being malignant as a function of the PARP1-positive tissue area (in percent) was estimated by nonparametric binary regression using the method of local likelihood.
PARPi-fl, in tissues both with and without prior DNA damage, was investigated as a probe for PARP1 imaging. It was shown that PARP1 expression in oral cancer is high, and that the uptake of PARPi-fl is selective, irrespective of whether cells were exposed to irradiation or not. It was also shown that PARPi-fl uptake increases in response to DNA damage, and that this increase is reflected in higher enzyme expression. These findings provide a framework for measuring exposure of cells to external beam radiation and for helping elucidate the effects of such treatments non-invasively in cancer subjects.
As described in the Background section, oral cancer is a type of malignant growth that more than 45,000 individuals will be diagnosed with in the United States in 2015 alone. Treatment options have improved over the last years, and the overall 5-year relative survival rates have increased from 52.7% in 1975 to 66.3% in 2007. This is in part due to the introduction of novel treatment options, one of which is intensity-modulated radiation therapy (IMRT). This type of radiation therapy allows the administration of ionizing radiation with varying intensities, effectively depositing DNA-damage events in a fairly defined region of the oral cavity. While IMRT is administered routinely, little is known about the spatial resolution of DNA damage on a case-by-case basis, and whether this damage can be visualized using injectable probes.
Although PARPi-fl was validated as an imaging agent for tumor tissue in the Examples described here, its use for tissues that underwent treatment has not been investigated. The Example provides: if PARPi-fl accumulates selectively in tumor nuclei, even after delivering a dose of radiation lethal to greater than 95% of a tumor cell population; if the marker is distributed and retained in tumor tissue, even after delivery of a therapeutic dose of radiation; and if PARP1 levels responds to ionizing radiation, and can this response be imaged using PARPi-fl.
The understanding of where, how, and to what extent radiation damage unfolds is critical to designing effective and optimized treatments regimens. The correlation between irradiation and DNA damage in oral cancer cells has been shown on the histological level, for example by measuring phosphorylated γH2AX foci formation. However, an injectable marker which can image such a response is to date an unmet clinical need.
It was shown herein that PARP1 is overexpressed in oral cancer. Using this model, it was determined in the present Examples that PARPi-fl is a selective marker in oral cancer cell lines, irrespective of whether they received ionizing radiation or not. The results described herein show that PARPi-fl uptake increases as a response to ionizing radiation within the first 48 hours. The results described herein also show that the elevated uptake correlates with higher PARP1 expression, and that uptake is selective not only in vitro, but also in vivo. Accordingly, PARP1 can serve as a marker of radiation-induced DNA damage.
PARP1 antigen expression was assessed in mouse tongue, FaDu and Ca127 xenografts using IHC to determine their basic PARP1 expression before irradiation. The staining was done using the Discovery XT processor (Ventana Medical Systems, Tucson, Ariz.). Paraffin-embedded formalin fixed 3 μm sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer (both Ventana Medical Systems, Tucson, Ariz.) and sections were blocked for 30 min with Background Buster solution (Innovex, Richmond, Calif.). The anti-PARP1 rabbit polyclonal antibody (sc-7150, Santa Cruz Biotechnology, Santa Cruz, Calif.) was incubated for 5 h (0.2 μg/ml), followed by 1 hour incubation with biotinylated goat anti-rabbit IgG (PK6106, Vector Labs, Burlingame, Calif.) at a 1:200 dilution. For detection, a DAB detection kit (Ventana Medical Systems, Tucson, Ariz.) was used according the manufacturer instructions. Sections were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific, Pittsburgh, Pa., USA). Incubating with a rabbit IgG instead of the primary antibody controlled for non-specific binding of the secondary antibody. Adjacent sections were stained with hematoxylin and eosin for morphological evaluation of the tissue. The staining was performed at the Molecular Cytology Core Facility of MSK. For quantification of PARP1 protein distribution, thresholding was performed (MetaMorph® Software, Molecular Devices, Sunnyvale, Calif.) on brown (PARP1) and blue (tissue) areas of digitalized sections and the relative PARP1 positive area was calculated by dividing the brown area by the total tissue area. 10 field-of-views were analyzed per section.
Cells were irradiated with 0, 2, 4, 6, 8 and 10 Gy in 75 cm2 culture flasks using a J.L. Shepherd Cesium irradiator (J.L. Shepherd, San Fernando, Calif.) at a dose rate of 174 cGy/min. Clonogenic survival was assessed. Briefly, after irradiation, cells were trypsinized, counted, and pre-defined numbers of viable cells were plated in 6-well plates in triplicate. In order to receive a sufficient colony count (e.g., from 50 and 100), two cell numbers were plated per irradiation dose (0 Gy: 200, 500; 126 2 Gy: 500, 1000; 6 Gy: 800, 3000; 8 Gy: 1600, 7000; 10Gy: 2500, 8000). Cells were cultured 10-14 days and then stained with 0.5% Crystal Violet (Sigma-Aldrich, St. Louis, Mo.) for 10 min at room temperature. Only colonies comprising at least 50 cells were counted, and a mean was calculated from the triplicate wells. The plating efficiency of each irradiation dose was calculated by dividing the number of counted colonies by the number of cells plated. The relative clonogenic survival was calculated by dividing the plating efficiency of a certain irradiation dose by the plating efficiency of untreated cells. Three independent experiments were carried out for each cell line.
To determine the binding of PARPi-fl to cells, cells were plated in 8-well Chamber Slides (Lab-Tek Brand; Nalge Nunc International, Naperville, Ill.). After 24 hours, cells were treated with 0 or 10 Gy irradiation in a J.L. Shepherd Cesium irradiator (J.L. Shepherd, San Fernando, Calif.) at a dose rate of 174 cGy/min. 24 hours post irradiation, cells were incubated with a 1 μM solution of PARPi-fl for 20 min at 37° C., followed by two 5 min incubations in full medium and one wash in PBS. Subsequently, cells were fixed with 4% Paraformaldehyde, plastic chambers were removed and slides were mounted with Mowiol® mounting medium containing Hoechst 33342 for counterstaining of cell nuclei. Imaging was done using a Leica SP5 upright confocal microscope (Leica, Buffalo Grove, Ill.), equipped with appropriate lasers and emission filters. PARPi-fl was imaged using the FITC channel and 488 nm laser excitation.
The change in PARPi-fl uptake was quantified in FaDu and Ca127 cells after irradiation using Flow Cytometry. First, cells were irradiated with 0, 2, 4 and 10 Gy in 25 cm2 culture flasks using the J.L. Shepherd Cesium irradiator (J.L. Shepherd, San Fernando, Calif.) at a dose rate of 174 cGy/min. At different time intervals post irradiation (6, 24 and 48 hours) PARPi-fl staining was initiated. Following a wash with PBS, cells were trypsinized, counted, and portions of 0.5×106 cells of the single cell suspension were aliquoted into 1.5 ml Eppendorf tubes (Eppendorf, Hamburg, Germany). For each time point and irradiation dose, samples were either left unstained, were stained with PARPi-fl or olaparib/PARPi-fl. Co-incubation with a 10-fold excess of the non-fluorescent PARP1 inhibitor olaparib was carried out to control for binding specificity of PARPi-fl. For staining, cells were washed with 1 ml FACS buffer (1% BSA (w/v) in PBS). Then, 1 ml of the staining solution (FACS buffer only, 0.5 μM PARPi-fl in FACS buffer or 5 μM olaparib/0.5 μM PARPi-fl in FACS buffer) was added for 20 min at 37° C., followed by one 5 min wash in 1 ml FACS buffer. Next, cells were centrifuged, the supernatant was aspirated and cells were re-suspended in 0.5 ml FACS buffer and transferred to 5 ml round bottom flow cytometry tubes (BD Biosciences, Bedford, Mass.) through a 40 μm strainer to remove doublets and left on ice until measurement in the flow cytometer (LSR® II, BD Biosciences, Bedford, Mass.). For each measurement, 10,000 events were counted. Raw data were processed in FlowJo software in order to calculate the changes in PARPi-fl uptake after irradiation. Cell clumps and debris were eliminated using the corresponding gates (forward and side scatter) for the unstained cell population. The gates were applied to all stained samples. PARPi-fl fluorescence was imaged in the FITC channel against side scatter (area).
Bilateral FaDu xenografts were inoculated 15 days before irradiation on the left and right side of the lower back of female athymic nude mice (n≥3/group). Tumor volume was measured with a caliper every 3-5 days and calculated by the formula π/6×(length×width×height of the tumor). Tumors on the right side were irradiated with 10 Gy using an image-guided microirradiator (X-Rad 225 Cx, Presicion X-Ray, North Branford, Conn.). The irradiation area was centered on the tumor by using the built-in cone-beam CT for soft tissue imaging and a 2×2 cm collimator. X-Ray irradiation was delivered at a dose rate of 3.1306 Gy/min while animals were under 2% isoflurane anesthesia.
At 24 hours and 48 hours after the irradiation, animals were sacrificed using carbon dioxide asphyxiation. Tumors were explanted, formalin-fixed and embedded in paraffin for immunofluorescent PARP1 staining. This was done following the protocol for IHC as described above, with the difference that detection was performed with Streptavidin-HRP D (from DABMap Kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 594 (T20935, Invitrogen, Carlsbad, Calif.), prepared according to the manufacturer's instructions. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and coverslipped with Mowiol® mounting medium (Sigma-Aldrich, St. Louis, Mo.). Immunofluorescence staining allowed for evaluation of the intensity of the PARP1 signal in each nucleus in addition to the PARP1 positive area. In each section, 10 fields of view were analyzed (total area 3.64 mm2). For each tumor, three sections were analyzed. Per group (irradiated and non-irradiated) 4 tumors were analyzed. The PARP1 quantification was done on digitalized slides using an automated segmentation and quantification protocol generated with the software MetaMorph® (Molecular Devices, Sunnyvale, Calif.) using the three scanned channels. The PARP1 positive area was determined by thresholding the red fluorescent area and dividing it by the whole tissue area, which was determined based on autofluorescence. PARP1 intensity was determined by measuring the red fluorescence intensity in all nuclei, which were thresholded using DAPI staining. The measured fluorescence intensities were averaged over all nuclei in each field-of-view.
Synthesis of PARPi-fl with BODIPY-FL as the Fluorophore
Synthesis of an example optical imaging agent PARPi-fl with BODIPY-FL as the fluorophore was carried out in a manner analogous to that previously described above. Briefly, the green fluorescent dye BODIPY-FL NETS-ester (Invitrogen, Carlsbad, Calif.) was conjugated to 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, followed by purification via preparative HPLC (Waters' XTerra C-18 5 μm column, 7 ml/min, 5% to 95% of acetonitrile in 15 min). PARPi-fl was obtained in 70-79% yield as a red solid in >97% purity. The identity of PARPi-fl was confirmed using ESI-MS (MS(+) m/z=663.4 [M+Na]+). For in vivo imaging studies, PBS (117 μl) was slowly added to an aliquot of PARPi-fl (50 μg, 75 nmol) in 50 μl of poly(ethylene glycol) (PEG300, Sigma-Aldrich, St. Louis, Mo.) to obtain a final injection volume of 167 μl.
Cohorts of subcutaneous FaDu tumor bearing animals were injected intravenously with PARPi-fl (75 nmol/167 μl PBS with 30% PEG300) 24 hours and 48 hours post irradiation and 90 min before sacrifice by carbon dioxide asphyxiation. Irradiated and non-irradiated tumors as well as tongues were explanted and the fresh tissues imaged immediately in the epifluorescence system IVIS (PerkinElmer, Waltham, Mass.) using the standard filter set for GFP imaging. Autofluorescence was removed using spectral unmixing. The PARPi-fl signal was analyzed semiquantitatively by measuring the average radiant efficiency [p/s/cm2/sr]/[μW/cm2] in regions of interest (ROIs) that were placed on the tissue under white light guidance. Resulting numbers are normalized for the integration time, binning, f/216 stop, field of view, illumination intensity, and the ROI area, making measurements comparable among each other. After epifluorescence imaging, the freshly excised whole tumors were imaged microscopically. Tissues were placed on a cover slip with a freshly cut surface facing the cover slip and images were taken on an inverted laser scanning confocal microscope using 488 nm laser excitation (LSM 5-Live, Zeiss, Jena, Germany). PARPi-fl stained tumors were also compared to tumors that did not receive PARPi-fl injection to assess the extent of autofluorescence in the images. To confirm the specificity of the PARPi-fl stain to PARP1 protein, cryosections of the excised tumors were stained with an anti-PARP1 antibody. For this, 10 μm cryosections were fixed with 4% Paraformaldehyde, blocked for 30 min with 3% goat serum, stained overnight with the primary antibody (rabbit anti-PARP1, 1 μg/ml, sc-7150, Santa 226 Cruz), rabbit IgG (isotype control) or antibody dilution buffer (no primary control, PBS containing 1%(w/v) BSA and 0.3% TritonX-100). This was followed by secondary antibody staining (goat anti228 rabbit-AF680, 2 μg/ml, Invitrogen). Slides were mounted with Mowiol (Sigma-Aldrich) containing Hoechst 33342 for nuclear counterstaining.
Statistical analysis was performed using GraphPad Prism 6. Unless otherwise stated, data points represent mean values, and error bars represent standard deviations of biological replicates. P values were calculated using a Student's unpaired t-test, corrected for multiple comparisons by the Holm-Sidak method with an alpha of 0.05 or 0.01 as the cutoff for significance.
Strong nuclear expression of PARP1 was observed in FaDu and Ca127 tumor tissue, but not in mouse tongue tissue (
Cell Survival and PARP1-fl Imaging after External Beam Irradiation
Before imaging the PARPi-fl uptake in response to external beam irradiation in HNSCC cell lines, the effect of irradiation was quantified on cell survival. Clonogenic assays revealed that cell survival decreased exponentially with increasing irradiation doses, as seen by reduction of the colony count (
PARP1 expression of FaDu and Ca127 cells was imaged using the fluorescent PARP1 inhibitor PARPi-fl. A quantitative relation between PARP1 expression and PARPi-fl binding was described above. PARPi-fl accumulated in the nuclei of FaDu and Ca127 cells, irrespective of the fact whether cells were irradiated with 10 Gy or not (
The effect of 10 Gy irradiation on PARP1 expression and PARPi-fl uptake was assessed in bilateral FaDu tumor bearing nude mice, where the tumor on the right flank was exposed to 10 Gy using an image-guided microirradiator on day 15 after tumor inoculation (
Tumor sections of irradiated and non-irradiated tumors were stained for PARP1 using Immunofluorescence staining at 24 and 48 hours post irradiation (
Determining the uptake of PARPi-fl macroscopically in freshly excised tumor tissues at 24 and 48 hours after irradiation, it was found that an increased uptake of PARPi-fl, mirroring the pattern of PARP1 protein expression (
To assess the influence of autofluorescence on the signal in the green fluorescence channel, a FaDu tumor, which had not received PARPi-fl, was also observed. Here, a low autofluorescence signal with a very narrow histogram, as opposed to the PARPi-fl containing tumor, which displayed a right shift of the histogram curve and much broader distribution of the fluorescent intensities, was observed (
Visualizing and quantifying the amount of external beam radiation delivered to a particular tissue compartment is a major challenge in radiation oncology research. Current methods used for determining the amount of radiation deposited in a given compartment largely rely on theoretical models and externally measured beam intensities. It has been recognized that such models have experienced considerable advances in past decades, but are also becoming prone to error with increasing complexity.
The Examples herein established a molecular imaging approach using rapid PARP1 targeted fluorescence imaging to yield a reproducible measure of the effects of external beam radiation to oral cancer tissue. The data shows that PARP1 is a robust biomarker, and that the agent accumulates selectively in OSCC cells both in vitro and in vivo, with and without previous irradiation treatment. It was also shown that PARP1 indeed responded to ionizing radiation, and that this change can be seen with PARPi-fl for both in vitro and in vivo experiments.
The disclosed imaging approach is based on the strongly elevated PARP1 expression in cancer tissue compared to its healthy surrounding host tissue. Specifically, the described xenograft mouse models showed that PARP1 expression was, with levels of 37.2±3.2% and 28.7±1.7% (for FaDu and Cal 27, respectively), 26-fold and 21-fold higher in tumor tissue than tongue tissue (1.4±0.4%,
While no changes in PARPi-fl uptake 6 hours after radiation exposure were seen, median PARPi-fl uptake changed at 24 hours, and particularly at 48 hours after an irradiation event. Changes were seen for as little as 2 Gy, and were more pronounced with increasing dose (
The increased expression of PARP1 post irradiation also provides for a combination of radiation therapy with PARP1 inhibitor therapy to mediate synthetic lethality to tumor tissue.
Accordingly, it was shown that PARP1 expression increases in response to external beam radiation, and that this increase can be observed in cell culture and on the tissue level. Further, the fluorescent imaging agent PARPi-fl is able to accumulate in irradiated cell nuclei of tumor tissues. Such accumulation indicates that PARP1 targeted imaging agents can be used to delineate tissues exposed to radiation. For examples, PARP1 targeted imaging agents can be used to elucidate the effects of changing perfusion, cell density and other architectural changes inside the tumor.
In other applications, PARP1 imaging can be applied to other modalities, for example whole body PET imaging, using 18F labeled or dual labeled (e.g., 18F and Bodipy-FL). PARP Inhibitors can be critical to enable clinical PARP1 imaging and a quantitative relationship between PARP1 expression in whole body PET imaging post irradiation and therapy outcome can be determined based on the disclosure herein.
Experiments were carried out using two human OSCC cell lines. FaDu (hypopharyngeal SCC; ATCC, Manassas, Va.) were maintained in MEM medium and Ca127 cells (tongue SCC; ATCC, Manassas, Va.) were maintained in D-MEM medium, both containing 10% (v/v) FBS and 1% Penicillin/Streptavidin. Cells were grown in monolayer culture at 37° C. in a 5% CO2 humidified atmosphere and passaged at 70-80% confluency.
Female athymic nude (NCr-Foxnlnu, Taconic, Hudson, N.Y.) were housed under standard conditions with water and food ad libitum. Animals were anesthetized with 2% isoflurane throughout tumor implantation, irradiation and imaging. To implement subcutaneous human OSCC tumors, 2×106 FaDu or Ca127 cells were dispensed in 50 μl medium, and 50 μl Matrigel™ (BD Biosciences, Bedford, Mass.) was added before injection on the lower back of the animals. For irradiation experiments, bilateral FaDu xenografts were used. Experiments were started 15 days after xenografting, when tumors had reached 100-150 mm3 volume. All animal experiments were done in accordance with institutional guidelines and approved by the IACUC of MSK and followed NIH guidelines for animal welfare. Animals were sacrificed before the experimental endpoint if tumors reached a volume of more than 1000 mm3, or animals displayed severe signs of distress such as rapid weight loss, crouching and impaired movement.
In certain embodiments, for PARPi-fl imaging, patients can first gargle a solution of PARPi-fl for 1 min, then spit out this solution and gargle with a cleaning solution (e.g., the solvent used for PARPi-fl) for 1 min. Then fluorescence imaging of the oral cavity and pharynx can be performed with an endoscope for approximately 10-30 min. The intensity and extent of the fluorescence signal can be recorded for the tumor and adjacent normal mucosa.
In certain embodiments, PARPi-fl is stored as lyophilized powder and is reconstituted within 1 h of application. The final concentration of the PARPi-fl can range between 100-1000 nM. The solvent can be 15% PEG300/15% sorbitol in 70% water.
In certain embodiments, fluorescence imaging can be performed with a multispectral fluorescence camera. The camera can be mounted on a short rigid endoscope that is routinely used for the clinical examination of OSCC patients. The camera comprises a charged coupled digital (EM-CCD) camera for sensitive fluorescence detection and two separate cameras for detection intrinsic fluorescence and color. This system allows to correct the fluorescence images for the autofluorescence of the mucosa and to overlay the corrected fluorescence images in realtime on the color (photographic/video) image. The system attains a variable field of view (FOV) of 15 cm×15 cm to 3 cm×3 cm with a corresponding resolution from 150 μm to 30 μm.
The intensity of the fluorescence signal in the tumor region and adjacent normal mucosa will be determined and documented on digital images. On the fluorescence images also the area considered as suspicious for tumor will be determined and compared in a descriptive way with the area considered as tumor on the non-fluorescent, color image of the tumor region.
In Phase I, patients in a first cohort can receive escalating concentrations of PARPi-fl (e.g., 100, 250, 500 and 1000 nM). If there are no dose limiting toxicities (e.g., local irritation, pain, systemic effects) in the three patients then the next cohort of three patients can receive next escalated concentrations of PARPi-fl. If there is at least one toxicity in the cohort of three patients then the concentration below this dose level can be recommended for Phase II concentration of PARPi-fl. This design follows the popular 3+3 design for finding the maximum patients if one toxicity is seen in the first set of three patients at a given dose level.
The reasons for this is two-fold: it is not anticipated that any toxicity at any of the concentration levels used and acceptable levels of toxicity for an imaging agent is much lower than that of a therapeutic. Despite the anticipation of no toxicity, the choice of the design (escalating levels with toxicity as the primary endpoint) reflected the general concerns of a first-in-man study. Furthermore, it is possible that at a certain level of concentration unspecific binding of PARPi-fl can decrease image contrast. Therefore, the ratio of tumor fluorescence to fluorescence in surrounding mucosa at each dose level can be measured. If the fluorescence ratio decreases from one level to the next higher one by more than 2.5 standard deviations of the lower level, the dose escalation can be stopped, and the level that produced higher contrast for the Phase II study can be used.
In phase II, using the concentration established in phase I, 18 additional patients can undergo the same imaging procedure as described above within 4 hours prior to planed tumor resection. Images can be recorded as for phase I and tumor-to-normal ratios calculated for the fluorescence signal. Areas on the image that have a fluorescence signal that is at least 2-times higher than in the contralateral mucosa can be marked as tumor.
Gold standard can be obtained through the pathologic analysis of the surgical specimen and each area marked as tumor by fluorescence imaging will have the corresponding gold standard obtained. Since imaging is performed within hours of surgery, it is not expected that patients will need to be replaced. In addition malignant areas in the surgical sample that were missed on the images can be found. Sensitivity can be estimated in the following way: number of areas identified as malignant by imaging divided by the total number of malignant lesions by gold standard. Confidence intervals for this will be estimated taking into account the multiple observations from each patient. With, for example, 18 patients and an average of two lesions per patient the confidence interval can be estimated to within +/−14% assuming a true sensitivity of 80% and within-patient correlation of 0.1 (in the absence of previous clinical studies, this number is based on the data from other imaging modalities).
Correlation between PARP1 expression and fluorescence signal can be estimated by rank methods using the fluorescence signal intensity, and the intensity of PARP1 staining on the areas where the intensity was obtained. Delineation of tumor infiltration by PARPi-fl imaging can be assessed by studying the fresh frozen samples under a fluorescence microscope, and the fluorescence from PARPi-fl can be compared with HE staining of an adjacent section (as described herein and, for example,
Table 3 shows exemplary PARP1 inhibitors that are binding to the same location (e.g. ABT-888, Abbott; AG014699, Pfizer; AZD2281, Astra-Zeneca; BMN-673, Biomarin; MK-4827, Merck). PARP1 imaging allows physicians to stratify patients in their appropriate treatment groups, enabling clinical decision making processes based on PARP1 levels.
This application is a continuation of U.S. patent application Ser. No. 15/565,369, filed Oct. 9, 2017, which is a National Stage Application of PCT/US2016/026717, filed Apr. 8, 2016, which claims the benefit of U.S. Application Ser. No. 62/145,873 filed on Apr. 10, 2015 and U.S. Application Ser. No. 62/291,463 filed on Feb. 4, 2016, the disclosures of each of which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant No. K25 EB016673-01 awarded by the NIH. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62145873 | Apr 2015 | US | |
| 62291463 | Feb 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16712919 | Dec 2019 | US |
| Child | 17846690 | US | |
| Parent | 15565369 | Oct 2017 | US |
| Child | 16712919 | US |
