Patent Application
20150224211
1. Field of the Invention
The present invention relates to targeted therapeutic methods for diagnosing and treating inflammation, infection, and/or cancerous tissue using leukocytes to deliver active agents to these foci.
2. Description of Related Art
Targeted, yet highly effective therapies for various diseases are intensively sought. For example, in cancer treatment, even though primary tumors can often be reduced or eliminated, the metastatic disease that subsequently occurs usually results in fatality. However, many of the recently developed targeted therapies are complicated, time-consuming, and expensive. In addition, there remains a need for targeted and effective therapies for infectious diseases, such as antibiotic-resistant infections, as well as various inflammatory conditions.
The present invention is broadly concerned with methods for the in situ treatment and/or diagnosis of infection, inflammation, and/or cancerous tissue in a subject having cancerous tissue or tissue infected or inflamed by a pathogen. The method uses naturally-occurring leukocytes of the subject (i.e., neutrophils, monocytes/macrophages, lymphocytes, and mixtures thereof) and comprises (consists essentially or even consists of) optionally administering a photosensitizing agent to the subject; administering a luminogenic substrate comprising luminol to the subject; and optionally detecting light generated by the luminogenic substrate to thereby image the infection, inflammation, and/or cancerous tissue. Advantageously, the naturally-occurring leukocytes accumulate in and near the infection, inflammation, and/or cancerous tissue and secrete oxidative species, these oxidative species react with the luminogenic substrate to generate light, which can not only be detected as noted above, but can also cause damage and destruction of the pathogen or cancerous tissue. In addition, the photosensitizing agent, when present, is activated by the light generated by the luminogenic substrate and the activated photosensitizing agent enhances the damage and destruction of the pathogen or cancerous tissue. Thus, the inventive methods involve in situ generation of light (energy), and preferably exclude photodynamic therapy utilizing external or externally-generated light sources (e.g., lasers, LEDs, etc. inserted into the subject).
Targeted methods of treating infection, inflammation, and/or cancerous tissue in a subject having cancerous tissue or tissue infected or inflamed by a pathogen are also described herein. The methods comprise (consist essentially or even consist of) providing naturally-occurring leukocytes of the subject (i.e., neutrophils, monocytes/macrophages, lymphocytes, and mixtures thereof); and loading the naturally-occurring leukocytes with an active agent. Advantageously, the loaded leukocytes accumulate in and near the infection, inflammation, and/or cancerous tissue and release the active agent to thereby treat the infection, inflammation, and/or cancerous tissue.
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The present invention is concerned with imaging lesions (e.g., tumors) for early detection and diagnosis of disease as well as treatment of infection, inflammation, lesions, and/or tumors by in situ photodynamic therapy. The present invention is also concerned with targeted methods of delivering active agents to sites of infection, inflammation, lesions and/or cancerous tissue. The methods of the invention are applicable in treating and/or diagnosing (theranostics) cancer, as well as infections, inflammation, and other types of lesions. The methods are suitable for diagnosing and/or treating subjects having cancerous tissue (e.g., tumors, metastases, and cancer cells) or tissue infected or inflamed by a pathogen (e.g., any infectious microorganism, such as a virus, bacterium, prion, or fungus). In the context of infection, inflammation, and disease, the invention is particularly advantageous for treating antibiotic-resistant pathogens.
In one or more embodiments, methods for the in situ treatment and/or imaging/detection of infection, inflammation, and/or cancerous tissue in a subject are described herein. Methods according to these embodiments take advantage of the naturally-occurring leukocytes of the subject. The term leukocytes, as used herein, encompasses neutrophils, monocytes/macrophages, lymphocytes, and mixtures thereof. Neutrophils are among the most numerous white blood cells in peripheral blood, and are often “first responders” to infection, inflammation, and other lesions. Monocytes in contrast to neutrophils are larger (hence could carry a larger therapeutic payload per cell). They are not as numerous as neutrophils but nevertheless can be isolated in large numbers from peripheral blood (or loaded after IV administration of drug-containing particles). Monocytes respond more slowly to infections than neutrophils, and differentiate into macrophages when they escape into tissues. Monocytes infiltrate tumors and chronic infectious foci such as granulomas, so they could be especially valuable as delivery vehicles for subjects suffering from such conditions. The term “naturally-occurring” as used herein denotes that the cells are in their natural form, and have not been: amplified, transformed, transfected, and/or otherwise modified such as through genetic or epigenetic modifications (e.g., have not been artificially altered to include a transgene and/or to change the expression, activity, or function of genes or gene products). In other words, the naturally-occurring cells are in their native, non-modified form, which may include naturally-occurring mutations, but does not encompass artificially-imposed genetic alterations. In one or more embodiments, the naturally-occurring cells have not been cultured (in vitro or ex vivo), where the term “cultured” refers to growing (expanding) cells, such as on a cell culture plate or in a culture tube. This is in contrast to “incubating” cells under conditions to maintain live cells without growing or expanding the cells. In one or more embodiments, such naturally-occurring cells may be autologous to the subject. In one or more embodiments, the naturally-occurring cells are “circulating” cells (in the bloodstream) of the subject, which means they have not been: injected into the subject, removed from the subject, cultured, and/or re-injected into the subject. In other words, methods according to the invention preferably exclude steps of administering non-naturally-occurring or otherwise exogenous leukocytes to the subject as a source of in situ light.
For imaging and in situ photodynamic therapy, the methods comprise administering a luminogenic substrate to the subject. The luminogenic substrate is a chemical that exhibits chemiluminescence, and more particularly is luminol (5-Amino-2,3-dihydro-1,4-phthalazinedione). Other luminogenic substrates are isoluminol (6-amino-2,3-dihydrophthalazine-1,4-dione), ABEI (6-((4-aminobutyl)(ethyl)amino)-2,3-dihydrophthalazine-1,4-dione) and L-0123 (8-amino-5-chloro-7-phenyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione), as well as acridinium derivatives, such as MMAC (9((4-methoxyphenoxy)carbonyl)-10-methylacridin-10-ium) or 2, -methoxy-10-methyl-9-(phenoxycarbonyl)acridin-10-ium (Yamaguchi et al, Analytica Chimica Acta 665 (2010) 74-78). The luminogenic substrate is administered locally or systemically via intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, intraarterial injection, or a combination thereof to deliver the luminogenic substrate to the subject's blood stream. The luminogenic substrate is typically administered dispersed in a pharmaceutically-acceptable carrier. The term “carrier” is used herein to refer to diluents, excipients, and the like, in which the luminogenic substrate may be dispersed for administration. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would naturally be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration. For example, compositions suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer. Exemplary carriers for use with the luminogenic substrate include complexes with beta-cyclodextrin in phosphate-buffered saline (PBS), or mixtures with polyethylene or oligoethylene in PBS, and the like.
Advantageously, the naturally-occurring leukocytes in the subject's bloodstream accumulate in and near any infection, inflammation, and/or cancerous tissue and secret oxidative species. Preferably, the naturally-occurring leukocytes accumulate in and near the infection, inflammation, and/or cancerous tissue within about 2 to about 5 days after administration of the luminogenic substrate. Due to this accumulation, these oxidative species are secreted in a targeted manner in and near the site of infection, inflammation, and/or cancerous tissue, where they react with the luminogenic substrate in the bloodstream to generate light that can have anti-cancer, anti-bacterial, anti-fungal, and/or anti-viral effects (i.e., causes damage and destruction of the pathogen or cancerous tissue). For example, myeloperoxidase and NADPH-oxidase generated by neutrophil secretion and/or apoptosis in and near the cancerous tissue results in the emission of light (bioluminescence). This is because luminol is oxidized by the neutrophil-generated oxidative species (superoxide, hydrogen peroxide, hypochlorite), creating an unstable species that breaks down emitting (blue) light. This blue light can be detected by appropriate imaging devices for early tumor detection and diagnosis. Thus, in one aspect, the method further comprises detecting the light generated by the luminogenic substrate to thereby image the site of infection, inflammation, and/or cancerous tissue. It can also be used to excite other fluorophores by either light absorption or fluorescence resonance energy transfer (FRET), resulting in a red-shift of emission. This in situ photodynamic therapy can be used for infectious disease or cancer treatment. Since human tissue is most transparent between 600 and 1000 nm, transferring the excited state energy to an appropriate fluorophore results in the capability of detecting the fluorescence occurring from deep-seated tumors.
For the photodynamic treatment of pathogens it is particularly preferred that a photosensitizing agent (aka “photosensitizer”) first be administered to the subject before administering the luminogenic substrate. Preferably, the photosensitizing agent is administered about 2 days before administering the luminogenic substrate. The photosensitizing agent can be administered locally or systemically via intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, intraarterial injection, or a combination thereof. Furthermore, the photosensitizing agent can be delivered as a prodrug by means of the same delivery modalities. The photosensitizing agent can be administered dispersed in a pharmaceutically-acceptable carrier as described herein, with preferred carriers for the photosensitizing agent being PBS, complexes of the photosensitizer or prodrug with alpha-, beta- or gamma-cyclodextrin, adsorbed onto albumin in PBS or dissolved PBS/polyethylene glycol mixtures. The photosensitizing agent is selected such that its absorption spectrum permits activation based upon the emission spectrum of the luminogenic substrate. In one or more embodiments, the luminogenic substrate generates light of a first wavelength, which activates the photosensitizing agent, when present. Accordingly, the photosensitizing agent generates or emits light of a second wavelength, which can be detected to determine the location of the infection, inflammation, and/or cancerous tissue in the subject. The activated photosensitizing agent can not only be used for imaging, but also enhances the damage and destruction of the pathogen or cancerous tissue. Exemplary photosensitizing agents include metallated (e.g. Mg(II), Zn(II), Pd(II), Pt(II)) and non-metallated porphyrins (e.g. protoporphyrin IX), dihydroporphyrins (chlorins), and tetrahydroporphyrins (bacteriochlorins), hypericin, and ruthenium-polypyridinium complexes, as well as photosensitizer-generating prodrugs (e.g., 5-aminolevulinic acid (“ALA”)), and mixtures or combinations thereof. Prodrugs that result in the formation of photosensitizing agents in vivo are particularly preferred. One example is ALA, a precursor for protoporphyrin IX. ALA is the substrate for the biosynthesis of protoporphyrin IX in the mitochondria of cancer cells and pathogens. Administration of ALA results in the formation of protoporphyrin IX in vivo, which acts as the photosensitizer in the photodynamic treatment process. Combinations of one or more photosensitizing agents can also be used.
Regardless of the embodiment, the damage and/or destruction to the cancerous tissue or pathogen from the photodynamic therapy (via the luminogenic substrate alone or in combination with the photosensitizing agent) preferably results in apoptosis of the cancerous tissue or pathogen, as opposed to necrosis.
In one or more embodiments, targeted methods of treating infection, inflammation, and/or cancerous tissue in a subject are also described herein. The methods use leukocytes to deliver an active agent to the site of inflammation, infection, and or cancerous tissue in the subject. Advantageously, the leukocytes are naturally-occurring and are selected from the group consisting of neutrophils, monocytes, lymphocytes, and mixtures thereof. In one or more embodiments, the leukocytes are autologous to the subject. The leukocytes are loaded with the active agent, and accumulate in and near the site of infection, inflammation, and/or cancerous tissue, where they naturally release the active agent (either by apoptosis or secretion) to thereby treat the infection, inflammation, and/or cancerous tissue in a targeted manner. The terms “load,” “loading,” or “loaded,” refer to the process or feature of the active agent being taken up by the leukocyte (such as through phagocytosis or other, endocytosis) and engulfed or internalized by the cell, such that it ends up inside the leukocyte (i.e., on the other side of the cell membrane).
In one or more embodiments, the naturally-occurring leukocytes are circulating leukocytes, which have not been removed from the subject, cultured, injected or re-injected into the subject. In other words, the active agent is loaded into the leukocytes in vivo (in the bloodstream) without removing the leukocytes from the subject. More particularly, loading in this aspect, comprises administering the active agent to the subject, where it is preferentially taken up by the circulating leukocytes in vivo. The active agent can be dispersed in a pharmaceutically-acceptable carrier as described herein, with preferred carriers for the active agent being a solution in PBS, a complex with alpha-, beta-, or gamma-cyclodextrin in PBS, or solution in PBS and polyethylene glycol. The active agent can be administered locally or systemically via intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, intraarterial injection, or a combination thereof. As the loaded leukocytes continue to circulate, they ultimately accumulate in and near the foci of inflammation, infection, and/or cancerous tissue to deliver their payload (i.e., the active agent).
In one or more embodiments, the leukocytes are loaded ex vivo. In other words, a blood sample is collected from the subject under ex vivo conditions, and the leukocytes are loaded outside of the subject's body (i.e., outside of the bloodstream), but without isolating them from the blood (for example, via an extracorporeal shunt). This removes the need to aseptically remove and culture cells without somehow changing them during culture. The fact that leukocytes in peripheral blood can be quickly loaded without isolating and culturing them is a significant advantage of this invention. Thus, the leukocytes are not isolated from the blood sample, and the blood sample containing the leukocytes is simply incubated with the active agent. In general, the active agent is incubated with the blood sample until the leukocytes in the blood sample are well loaded. It will be appreciated that specific incubation times will vary depending upon the target leukocytes to be loaded. That is, neutrophils take up the active agents more quickly than other leukocytes, such as monocytes or lymphocytes. Thus, shorter incubation times are required for neutrophils, as opposed to other leukocytes. In general, the active agent will be incubated with the blood sample for less than about 12 hours, preferably from about 30 min to about 6 hours, and more preferably from about 30 minutes to about 1 hour.
Regardless, the leukocytes in the blood sample take up the active agent, and then the blood sample containing the loaded leukocytes is administered to (i.e., injected into) the subject. In one or more embodiments, the blood sample containing the loaded leukocytes can be dispersed in a pharmaceutically-acceptable carrier for administration to the subject. Although the inventive method is intended primarily for autologous leukocyte injections, it is contemplated that a blood sample collected from a first subject could be loaded ex vivo and then injected for targeted therapy into a second subject (e.g., who is otherwise a compatible recipient of such a blood sample). Regardless, the loaded leukocytes ultimately accumulate in and near the foci of infection, inflammation, and/or cancerous tissue and release the active agent to thereby treat the infection, inflammation, and/or cancerous tissue in the recipient subject. Preferably, the blood sample containing the loaded leukocytes is administered to (i.e., injected into) the subject less than about 24 hours after the blood sample has been collected, and more preferably less than about 12 hours after collection.
Regardless of the embodiment, it will be appreciated that the treatment methods of the invention are fast, highly efficient and extremely targeted drug delivery methods for lesion-causing diseases, including infectious diseases, inflammation, and cancer. These drug-delivery methods utilized lesion-homing leukocytes, which are abundant in peripheral blood. Autologous cells can also be used to avoid immune rejection.
In one or more embodiments, the active agent is loaded into the leukocytes using a delivery vehicle in which the active agent is encapsulated. The delivery vehicle will typically comprise a targeting moiety on the surface thereof for preferential uptake by the naturally-occurring leukocytes. This is particularly advantageous for in vivo loading of the active agent. Exemplary targeting moieties includes peptides, antibody fragments, and negative surface charges mimicking bacteria, and the like that are specific to one or more of the neutrophils, monocytes, and/or lymphocytes, and can be attached to the surface of the delivery vehicle for preferential uptake. In one or more embodiments, the delivery vehicle is selected from the group consisting of liposomes, polymersomes, supramolecular structures, vesicles, exosomes, and combinations thereof. The active agent is incubated with the delivery vehicle until encapsulated in the delivery vehicle (and preferably for about 1 hour to about 12 hours, and more preferably for about 1 to about 6 hours). The preloaded delivery vehicle can then be used to load the active agent into the leukocytes in vivo or ex vivo.
In one or more embodiments, the delivery vehicle is a non-pathogenic, inactivated bacteria. That is, the leukocytes can be quickly loaded via incubation with non-pathogenic, chemical or heat-inactivated bacteria that have been preloaded with the active agent(s). For example, loading leukocytes with traditional delivery vehicles (e.g., liposomes) outside the bloodstream should be done rapidly, given that leukocytes can only survive up to a maximum of 24 hours ex vivo. However, incubation in whole blood with pre-loaded, killed bacteria can be accomplished much more quickly enhancing viability of the leukocytes for ex vivo loading. For example, neutrophils can be loaded with pre-loaded bacteria in about an hour. In the method, the bacteria are first loaded with the active agent by culturing the bacteria under appropriate conditions with the active agent, and then the bacteria are heat or chemically inactivated. Blood is drawn from the subject, and the preloaded, inactivated bacteria are incubated with the peripheral blood sample (containing the unisolated leukocytes) until the leukocytes are well-loaded. As noted above, specific incubation times will vary depending upon the target leukocytes to be loaded. In general, the inactivated bacteria are incubated with the peripheral blood sample is incubated less than about 12 hours (preferably about 30 minutes to about 2 hours, and more preferably from about 30 min to about 1 hour). For neutrophils, the incubation time is preferably about 1 hour. Any free (unloaded bacteria) is then separated from the loaded leukocytes. For example, the blood is centrifuged to remove free bacteria, the pellet is re-suspended in phosphate buffered saline (PBS) or other media, then administered to (i.e., injected into) the subject. Monocytes can be loaded in similar fashion as described for neutrophils, except that incubation times would be longer since monocytes take up bacteria more slowly. In other words, monocytes still would not have to be separated or isolated from blood, but a centrifugation or magnetic separation step would be used to separate unloaded bacteria from the cells prior to re-infusion of the loaded cells into the subject.
Non-pathogenic, inactivated bacteria can also be used to load the leukocytes in vivo. In one or more embodiments, the bacteria can be opsonized before administering the loaded bacteria (via a pharmaceutically-acceptable carrier) to the subject for preferential uptake by the naturally-occurring leukocytes in vivo. In one or more embodiments, the preloaded bacteria is mixed with bacteria-specific antibodies, and this mixture is administered to the subject for preferential uptake by the naturally-occurring circulating leukocytes in vivo.
The approach of using bacteria as the delivery vehicle for loading the active agent into the leukocytes is suitable for use with most non-pathogenic bacteria strains. Non-limiting examples of suitable bacteria include those selected from the group consisting of Magnetospirillum, Lactobacillus, Micrococcus luteus, Escherichia coli (e.g., laboratory strains used for plasmid isolation), and the like.
A wide variety of active agents can be used in the inventive methods. Thus, the term “active agent” as used herein refers to any therapeutic or diagnostic agent capable of being loaded into a leukocyte. Non-limiting examples of various types of agents that one might desire to deliver to a site of inflammation, infection, and/or cancerous tissue, include anticancer or chemotherapeutic drugs, antimicrobial drugs, anti-fungal drugs, anti-viral drugs, and/or anti-inflammatory drugs. Thus, suitable active agents include small molecule drugs, chemotherapeutic drugs (e.g., doxorubicin, salinomycin, SN-38, cuculinin), fluorophores, photosensitizers (e.g., protoporphyrin IX, hypericin), antimicrobial agents (e.g., antibiotics, antibacterial agents, anti-viral agents, or anti-fungal agents, such as chlorhexidine, betadyne, gentamycin, tetracycline), anti-inflammatory agents (e.g., non-steroidal anti-inflammatory drugs, corticosteroids, curcumin, sulforaphane, and mixtures thereof), matrix metalloproteinase (MMP) inhibitors, MDR blockers (e.g., verapamil, resveratrol), biologics, and/or magnetic nanoparticles. In one or more embodiments, for even more targeted delivery, the active agent can also be tethered to nanomaterials such as dextran or dendrimers or magnetic nanoparticles by a caspase-cleavage sequence for targeted release only in the vicinity (proximate region) of the target tissue upon apoptosis of the leukocyte. These tethered active agents delivered via the leukocytes can serve as powerful hidden prodrugs to be released when the leukocytes undergo apoptosis. That is, the natural fate of leukocytes is to undergo apoptosis shortly after they have reached the foci of inflammation, infection, and/or cancer. Likewise, caspases are activated only by the process of apoptosis. Thus, the tethered drug will be released in active form only when the leukocytes undergo apoptosis and the tether is cleaved by the activated caspases. Hence, this is a highly specific way to deliver a powerful drug as an inactive prodrug specifically to a tumor or site of infection or inflammation using the lesion-homing leukocytes.
In one or more embodiments, the delivery vehicle itself can be chosen as being integral to the active agent. In particular, bacteria that make magnetic nanoparticles naturally (e.g., Magnetospirillum, ATCC 700264) can be used to load magnetic nanoparticles into the leukocytes, in order to generate cell-mediated magnetic hyperthermia in the tumors. These bacteria can be heat inactivated after making the magnetic nanoparticles and loaded into leukocytes for cell-mediated magnetic hyperthermia. Of course, cell-mediated magnetic hyperthermia can also be achieved by loading magnetic nanoparticles into the leukocytes via another suitable delivery vehicle (e.g., other bacteria or traditional delivery vehicle).
The targeted delivery methods can also be used to deliver a photosensitizer (e.g., protoporphyrin IX, hypericin, etc.), into a tumor for an alternative photodynamic therapy technique. Many photodynamic drugs can also red-shift the emitted light that would concurrently allow imaging of tumors deeper within the body, thus permitting theranostic applications. For example, the loaded leukocytes of the invention can also be used for in situ photodynamic therapy of cancer or microbial infections. In one embodiment of the invention, leukocytes are loaded with fluorophores that are released in active form when the leukocytes undergo apoptosis, shortly after reaching a lesion. Leukocyte-generated oxidative species cause bioluminescence of fluorophores, which can detected by appropriate imaging devices and can be utilized for early, deep-seated tumor detection.
It will be appreciated that the embodiments described herein utilize leukocytes, as natural homing devices for targeted therapy of cancer or infectious lesions. These abundant (preferably autologous) cells can be specifically loaded with an effective amount of therapeutic or diagnostic agents using delivery vehicles having targeting moieties attached thereto that are specific for the leukocytes, to create a stealth system for delivering therapeutic drugs such as for anti-cancer, anti-microbial and anti-inflammatory applications, hidden prodrugs for release at targeted sites, and cancer therapy for deep and disseminated tumors and cell-mediated magnetic hyperthermia. It will be appreciated that the “effective amount” of the agents will depend upon the particular agent used and the subject. In general an “effective amount” refers to the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic effect as against the inflammation, infection, and/or cancerous tissue. Once the leukocytes are in the vicinity of the target tissue (tumor, lesion, etc.) the leukocytes undergo natural apoptosis, releasing their payload (i.e., the active agent). In the case of cancer, it will be appreciated that clinically relevant agents (e.g., DMXAA (5,6-Dimethylxanthenone-4-acetic Acid, ASA404, Vadimezan), cytokines, G-CSF, etc.) can be co-administered along with or prior to the loaded leukocytes to increase migration into tumors. Alternatively, a therapy causing necrosis in the tumor will increase infiltration of leukocytes.
The inventive methods offer minimal risk of immune rejection during cancer therapy, as autologous neutrophils are the first responders to tumors and other lesions, and the patient's own leukocytes can be used for the theranostic procedure.
It will be appreciated that therapeutic methods described herein are applicable to human subjects as well as any suitable non-human subject, including, without limitation, dogs, cats, and other pets, as well as, rodents, primates, horses, cattle, pigs, etc. The terms “therapeutic” or “treat,” and the like, as used herein, refer to processes that are intended to produce a beneficial change in an existing condition (e.g., infection, disease, condition) of a subject, such as by reducing the severity of the clinical symptoms and/or effects of the inflammation, infection, and/or cancer, and/or reducing the duration of the infection/symptoms/effects in the affected subject. The methods can be also applied for clinical research and/or study. Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.
As used herein, the phrase “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting “greater than about 10” (with no upper bounds) and a claim reciting “less than about 100” (with no lower bounds).
The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
We tested the hypothesis that neutrophils secreting myeloperoxidase into tumors could be exploited to generate a light source that could be used for PDT. 1×105 4T1 mouse mammary carcinoma cells (ATCC) were transplanted into a mammary fat pad of a mouse. Four, six, eight and ten days after transplant, luminol (Sigma-Aldrich) was administered intrapertioneally (IP)(250 mg/kg) and the mouse was imaged on an IVIS Lumina imaging apparatus. The results are shown in
We have tested whether tumor-tropic neutrophils can be efficiently loaded without separation from the blood using liposomes with targeting peptides on their surface. The protocol is outlined below.
We have tested whether neutrophils loaded with non-pathogenic bacteria carrying doxycycline (an antibiotic) can be more effective than neutrophils alone in eliminating bacteria, and have found that this is indeed the case (
In some embodiments, an in vitro test can be carried out to show that neutrophils loaded with antibiotic-filled lacobacilli can reduce numbers of bacterial colonies, as shown in
The protocol for bacterial enumeration following treatment with PMNs is outlined below.
This protocol has been repeated using chlorhexidine (an example of a broad spectrum antiseptic) and Micrococcus luteus, which is a gram positive, spherical bacteria that inhabits soil, dust, water and is part of normal human skin flora. This process is illustrated in
To determine the retention of CHX in the bacteria, samples were placed on a shaker for 7 days and the supernatant was measured to determine the amount of CHX that has left the cells. The results are shown in
The experiment was repeated with results being shown in
In a preliminary safety study, BALB/c mice (10-wk old; total 6) were injected intravenously (tail vein) with one million syngeneic neutrophils loaded with an average of 20 cells of M. luteus modified with either 1% or 2% chlorhexidine gluconate (CHX) (CHX microparticles). The animals were observed for 5 days post challenge to determine whether they demonstrated any clinical signs of toxicity. At the end of 5 days, the mice were euthanized and their kidneys, liver, brain, lung, spleen and heart were fixed in 10% formalin for histopathological evaluation. After clinical observation for one week, mice were euthanized. No gross or microscopic lesions were present in the liver, spleen, kidneys, heart, skeletal muscles, pancreas, brain or the site of intravenous administration. Spectroscopic analysis of the mouse liver revealed no residual CHX.
In an efficacy study that followed, Fusobacterium necrophorum subsp. necrophorum strain 8L1 was grown overnight from a single colony in PRAS-BHI. 0.3 ml of the starter culture was added to 10 ml of fresh PRAS-BHI broth and grown to an O.D.600 of 0.7. 1 ml of this culture was diluted 1:40 to achieve a final concentration of approximately 4·106 CFU/ml. 400 μl of the diluted bacteria was injected intraperitonially into 10 week old BALB/c mice. The mice were observed for two days before they were treated with neutrophils carrying antimicrobial cargo or controls.
Ten-week old BALB/c mice were randomly assigned into 6 groups with 9 mice per group. On day zero, all mice were infected intraperitonially with an infectious dose (approximately 1×107 CFU) of F. necrophorum. On day 3, mice were injected (via tail vein) with 100 μl PBS (control), unmodified neutrophils, neutrophils carrying microparticles (an average of 20 cells of M. luteus loaded with chlorhexidine), or neutrophils containing unmodified, heat deactivated M. luteus. The animals were monitored for clinical signs for 5 days after treatment and were euthanized if any clinical signs developed. The mice were euthanized 5 days post treatment and all mice were examined post mortem for abscesses in the livers. Livers of the mice were weighed and homogenized in a tissue homogenizer for 1 min in modified lactate (ML) broth.
While none of the mice treated with neutrophils carrying CHX developed liver abscesses, at least two mice in the other groups developed gross or microabscesses. The most severe was the PBS only group, where 5 out of 9 mice developed abscesses. F. necrophorum bacterial load in the liver homogenate was significantly lower in mice treated with neutrophils loaded with microparticles containing CHX (
The present application is a continuation-in-part of PCT/US2013/070515, filed Nov. 18, 2013, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/728,083, filed Nov. 19, 2012, entitled NEUTROPHILS AS DELIVERY CELLS FOR IMAGING AND DISEASE THERAPY, each of which are incorporated by reference in its entirety herein.
This invention was made with government support under #NSF-CBET 933701 and #NSF-DMR 1242765, awarded by the National Science Foundation. The United States government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61728083 | Nov 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2013/070515 | Nov 2013 | US |
| Child | 14694511 | US |
