USING SAME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 62/697,206, filed on July 12, 2018 which application is incorporated by reference herein in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure provides, inter alia, a fluorescent probe that binds specifically to vesicular monoamine transporter type 2 (VMAT2), which is suitable for live cell imaging and the identification of immature beta cells as well as the characterization of heterogeneous populations. Non-invasive methods for morphometrically and quantitatively analyzing beta cells in a subject that are useful in the management of metabolic disorders including diabetes are also provided herein.

BACKGROUND OF THE DISCLOSURE

[0003] The islet b-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand (Henquin, 2011 ). In addition to glucose, fatty acids and gut derived peptides, net insulin production is regulated by a number of other molecules, including a number of classical neurotransmitters including dopamine. It has been reported that b-cell secreted dopamine (DA) mediates a glucose stimulated insulin secretion (GSIS) inhibitory circuit in human b-obIIe^ίίhreoh et al., 2012, Ustione and Piston, 2012, Raffo et al., 2008). Using cadaveric human islets in vitro, it has been demonstrated that islet b-cells co-secrete insulin and dopamine in response to glucose stimulation , both in vivo and in vitro, and that dopamine down regulates insulin secretion via dopamine type 2 like-receptors (D2R) expressed by b-cells. In this circuit, DA, synthesized de novo or imported by dopamine (reuptake) transporter (DAT), is stored in b-cell vesicles by the action of the vesicular monoamine transporter type 2 (VMAT2). Thus, in the pancreas, VMAT2 is a central control point for regulation of insulin secretion and analogous to its role in the CNS of regulating monoamine neurotransmission (Wimalasena, 2011 ).

[0004] The function of VMAT2 in insulin secreting beta cells is as follows 1 ) DA and/or LDOPA are produced at a distant organ (i.e. , gut) and travel via the circulation to the b-cells, 2) L-DOPA is transformed to DA (by DOPA decarboxylase or AADC) within the b- cells , 3) DA is taken up by DAT in the b- cells, 4) DA is concentrated by the actions of VMAT2 for vesicular storage in b-cells and released in high concentration near DA receptors similar to that reported for the synapse. The insulin granules also contain D2R. During GSIS, DA and insulin are released and D2R is delivered to the cell surface where it binds DA. DA signaling through D2R is a powerful inhibitor of glucose dependent insulin secretion (Ustione et al. , 2013).

[0005] VMAT2 is tissue restricted marker of beta cells in the pancreas, a characteristic that has been leveraged to perform PET imaging of human beta cells in situ using [18F] or [11 C] labelled dihydrotetrabenazine (Souza et al., 2006, Freeby et al., 2016, Goland et al., 2009). Dihydrotetrabenazine (DTBZ) is a VMAT2 ligand with a nanomolar affinity constant (Scherman et al., 1983). On this basis, the present disclosure is directed to develop a VMAT2 ligand with a fluorescent reporter, suitable for live cell imaging and demonstrate its utility in quantification of living beta cell vesicle maturation.

SUMMARY OF THE DISCLOSURE

[0006] In one embodiment, the present disclosure is a probe that binds VMAT2 with high specificity and affinity. The probe is comprised of, e.g., a small molecule known to bind to VMAT2, and a detection agent, such as, e.g., a fluorescent molecule that does not interfere with the interaction between the probe and VMAT2. This probe binds with high affinity to cells expressing VMAT2, and can therefore be used to identify, image, track, and isolate beta cells, including immature beta cells. The present disclosure can probe heterogeneous populations of beta cells, and also has broad applications in basic research, drug discovery, and diagnostics.

[0007] In another embodiment, the present disclosure is a compound (Dansyl dihydrotetrabenazine, DDTBZ) having the following structure:

[0008] In another embodiment, the present disclosure is a vesicular monoamine transporter type 2 (VMAT2) binding probe comprising a compound disclosed herein.

[0009] In yet another embodiment, the present disclosure is a kit comprising the probe disclosed herein together with instructions for its use.

[0010] In a further embodiment, the present disclosure provides a method for determining the maturity of a beta cell in the pancreas of a subject. This method comprises:

a) obtaining and culturing beta cells from the subject; b) staining the beta cell culture with DDTBZ and at least one additional fluorescent agent; c) obtaining at least one computerized image of the stained beta cell culture; and d) morphometrically analyzing the computerized image in order to determine the maturity of the beta cell.

[0011] In yet another embodiment, the present disclosure provides a method for diagnosing a metabolic disorder in a subject. This method comprises:

a) obtaining beta cells from the subject and culturing with a moderate glucose concentration basal medium; b) staining the beta cell culture with DDTBZ and FluoZin-3 AM; c) obtaining at least one computerized image of the stained beta cell culture; d) morphometrically and quantitatively analyzing the computerized image to determine the average vesicle diameter of the cultured beta cells; e) exchanging at least a portion of the culture medium in step (a) with a low glucose concentration basal medium and incubating; f) repeating steps (b) to (d) with the cultured cells from step e) to determine the average vesicle diameter of the cultured beta cells; and g) if the average vesicle diameter of the beta cells determined in step (f) is not significantly reduced relative to the average vesicle diameter of the beta cells determined in step (d), it is indicative of the presence of a metabolic disorder in the subject.

[0012] In another embodiment, the present disclosure provides a method for identifying the presence of immature beta cells in the pancreas of a subject. This method comprises:

a) administering an effective amount of DDTBZ to the subject; b) obtaining at least one computerized image of at least a portion of the pancreas of the subject; and c) quantitatively analyzing the computerized image in order to identify the presence of immature beta cells in the pancreas of the subject.

[0013] Still another embodiment of the present disclosure is a compound (iodo- DDTBZ) having the following structure:

[0014] Yet another embodiment of the present disclosure is a method for determining the maturity of a beta cell in the pancreas of a subject, comprising:

a) obtaining and culturing beta cells from the subject; b) staining the beta cell culture with a radioactive compound disclosed herein; c) obtaining at least one computerized image of the stained beta cell culture; and d) morphometrically analyzing the computerized image in order to determine the maturity of the beta cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The application file contains at least one photograph executed in color. Copies of this patent application with color photographs will be provided by the Office upon request and payment of the necessary fee.

[0016] Fig. 1 shows the synthesis of dansyl dihydrotetrabenazine (DDTBZ). Synthesis of 2-hydroxy-3-isobutyl-9-methoxy-1 ,3, 4, 6, 7, 1 1 b-hexahydro-2H-pyrido[2, 1 - a]isoquinolin-8-yl 5-(dimethylamino)naphthalene-1 -sulfonate ((+) DDTBZ) reagents and conditions: (a) 5-(dimethylamino)naphthalene-1 -sulfonyl chloride (dansyl chloride), N,N-dimethylpyridin-4-amine (DMAP), dichloromethane (DCM), 0 °C, room temperature. [0017] Fig. 2 shows some dihydrotetrabenazine based structures. 1 ) Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-methoxy-10-methoxy-1 ,2, 3, 4, 6, 7,- hexahydro-11 bH-bezo[alpha]-quinolizine) (DTBZ). 2) (+)-2-Hydroxy-3-isobutyl-9-(3- fluoropropoxy)-10-methoxy-1 ,2,3,4,6,7-hexahydro-11 bH-benzo[a]quinolizine

(FPDTBZ). 3) 2FI-Benzo[a]quinolizine-2,9-diol, 1 ,3, 4, 6, 7,11 b-hexahydro-10-methoxy- 3-(2-methylpropyl)-, (2R,3R, 11 bR)

[0018] Fig. 3 shows the excitation and emission spectra of DDTBZ. Stock solutions of DDTBZ in DMSO were diluted to 20 mM and their excitation-emission spectra recorded. Results are diluent (PBS, 1 % DMSO) background subtracted.

[0019] Fig. 4 shows that DDTBZ binding colocalizes with mCherry-VMAT2 and binds preferentially to VMAT2 transfected cells. Successive z focal planes of a HEK- DAT mCherry-VMAT2 cell stained with DDTBZ ( Panel A-D and E-H). DDTBZ (75 mM) was added to cell cultures, cells were incubated and then washed and imaged (excitation at 385 nm, emission collected at 465-525 nm) (Panel A and E). The VMAT2-m Cherry fusion protein was visualized at 645-720 nm (Panel B and F). Panels C and G are the colocalization plots for the data collected in Panels A and B, and Panels E and F. The vertical axis represents pixel intensity for the magenta channel, the horizontal axis represents the pixel intensity for the green channel. The z axis is the heat map scoring of the number of pixels with identical intensities. The pixel intensities in each channel laying to right of each plot were significantly correlated as indicated by their Pearson's correlation coefficients ( 0.89, Panel C, 0.79, Panel G) and are shown in white in the pseudocolor images. The pseudocolor merged images of panel A plus B and E plus F appear in Panels D and FI, respectively. Panel I, transillumination image of untransfected FIEK 293 cells. Panel J, same field as Panel G imaged with the same exposure parameters as Panels A,B,E, and F. Scale bar represents 10 mM.

[0020] Fig. 5 shows that vesicular binding of (+) DDTBZ distinguishes VMAT2 positive cells from the non-specific binding pattern in VMAT2 negative cells. FIEK293, PANC1 and 5637 cells were stained with (+) DDTBZ, Nuclear Mask Deep Red (NMDR) and Lysotracker Red DND-99 (LT). The (+) DDTBZ signal was acquired with a 495BP30 filter and is shown in green in the pseudocolour merged images at right. NMDR and LT signals were imaged together in a 655 to 710 nm window and are shown in red in the pseudocolour merged images at right. Scale bar represents 10 microns. [0021] Fig. 6 shows the specificity, displaceability and saturability of DDTBZ. HEK-DAT-mCherryVMAT2 or untransfected HEK 293 cells were cultured in 96 well glass bottom black plastic plates. DDTBZ (20 mM) was added to the cultures and the bound fluorescence quantitated. Results are the averages of three independent experiments. The IC50 (DDTBZ by DTBZ « 7 pM) was estimated graphically (dotted line). Error bars are the standard error of the mean.

[0022] Fig. 7 shows that DDTBZ, NeuroSensor 521 and LysoTracker® Red DND- 99 staining of live human beta cell cultures identifies VMAT2 positive, monoamine positive, acidic vesicles in a morphologically diverse population of cells. Cell cultures were stained with the three probes and imaged in their corresponding channels. A merged pseudocolor image (right panels) represents the three grey scale images translated to an RGB image. Scale bar represents 10 pM.

[0023] Fig. 8 shows that DDTBZ, FluoZin™-3 AM zinc probe and Lysotracker staining identify a morphologically heterogeneous population of cells with VMAT2 positive, Zinc positive acidic vesicles. A merged pseudocolor image (right panels) represents the three grey scale images, (+) DDTBZ (blue channel), FluoZin-3 AM (green channel) and Lystotracker Red DND-99 (red channel) translated to an RGB image. These are representative images from identical experiments performed on three donors (141 , 212 and 556). Scale bar represents 10 pM.

[0024] Fig. 9 shows the insulin secretion characteristics of b-cell cultures. Panel A b-cell cultures were cultured in regular media containing 12 mM glucose for 5 days (Hi Glue), then assayed for glucose stimulated insulin secretion (GSIS). Panel B b- cell cultures were cultured in regular media containing 12 mM glucose for 5 days, transferred to media supplemented to 4 mM glucose (Lo Glue), then assayed for GSIS. The insulin content of supernatants was measured by ELISA and normalized to the DNA content of the well. Asterisks show statistical significance (p < 0.05, paired t test) of the difference between basal and stimulated insulin secretion. Donor RRID appears within each symbol.

[0025] Fig. 10 shows the frequency distribution of vesicle diameters in human beta cells cultures from three donors. The OpenCFU outputs of vesicle counts and diameters were binned (in intervals of 90 nm starting at 210 nm and ending at 2010 nm) and the frequency distribution histograms prepared. Automated vesicle counts ranged from 300-800 per cell. Panel A-C, the mean vesicle diameters (nm) and t test significance of the difference between the populations are shown in the legend. Panel D, The average vesicle diameter for cells (n=37) in a 12 mM glucose beta cell culture obtained from donors 1 islets was calculated a presented as a histogram.

[0026] Fig. 1 1 shows that frequency distribution of vesicle diameters in human b- cells cultures from three donors varies according to culture glucose concentrations. The OpenCFU outputs of vesicle counts and diameters were binned (in intervals of 100 nm starting at 250 nm and ending at 2500 nm) and the frequency distribution histograms prepared. Automated vesicle counts ranged from 300-800 per cell, total measurements ranged from about 9k to 15 k vesicles per culture. Experiments performed on three different healthy donors and one donor diagnosed with T2D (085, 952, 585 and 184(T2D)).

[0027] Fig. 12 shows that a subset of cells in islet cultures do not bind stain well for (+) DDTBZ, NeuroSensor 521 or FluoZin-3 AM yet stain for LysoTracker® Red DND-99. Cell cultures were stained with the three probes and imaged in their corresponding channels. Images obtained from two cell cultures from islet donors 025 and 141. The merged pseudocolour image (far right panels) represents the three grey scale images, (+) DDTBZ (blue channel), NeuroSensor 521 or FluorZin-3 AM (green channel) and Lystotracker Red DND-99 (red channel) translated to an RGB image. White arrows identify (+) DDTBZ and FluorZin-3 AM weak/negative cells. Scale bar represents 10 pm.

[0028] Fig. 13 shows that the bT0-6 murine cell line is heterogeneous with respect to vesicular binding of (+) DDTBZ. bT0-6 were stained with (+) DDTBZ, Nuclear Mask Deep Red and Lysotracker. The (+) DDTBZ signal was acquired with a 495BP30 filter and is shown in green in the pseudocolour merged images at right. Nuclear Mask Deep Red and LysoTracker Red DND-99 signals were imaged together in a 610 to 700 nm window and are shown in red in the pseudocolour merged images at right. White arrows indicate vesicles staining with (+) DDTBZ. Scale bar represents 10 microns.

[0029] Fig. 14 shows inhibition of (+) DDTBZ vesicular fluorescence by (±) DTBZ. Fluman islet b cell cultures were stained with (+) DDTBZ and the indicated concentration of racemic (±) DTBZ for 45 minutes at room temperature and then washed. Cultured cells were then imaged at 100 x. Twenty to 40 cells (corresponding to > 1000 vesicles) per condition were imaged with the following filter set Excitation 330WB80, Dichroic 400DCLP, Emission 495BP30. Cellular vesicles were identified by OpenCFU software and vesicular signal intensity was calculated for each vesicle as the quotient of the mean pixel intensity and vesicle pixel area. Mean vesicular signal intensities for each condition and the percent inhibition relative to cultures treated only with (+) DDTBZ were calculated. All reported values were significantly different (p<0.05) from one another. Results obtained from a single islet donor (567).

[0030] Fig. 15 shows that DDTBZ, NeuroSensor 521 and LysoTracker® Red DND-99 staining of live porcine beta cell cultures identifies VMAT2 positive, dopmine positive, acidic vesicles in a population of dispersed islet cells. Cultures were stained (Panels left to right) with DDTBZ, NeuroSensor 521 and LysoTracker® Red DND-99 and imaged in the blue, green and red channels as described in Fig. 7. Scale bar in cropped image represents 10 mM.

[0031] Fig. 16 shows that insulin and proinsulin colocalize with VMAT2 in the vesicles of cultured human beta cells. Cultures of human islet cells were fixed for immunohistochemistry with stained with anti-insulin, anti-proinsulin and anti VMAT2 antibodies. Nuclei were counterstained with DAPI and imaged. The far right panels are the merged pseudocolor images from the single focal plane obtained from photographs prepared in each channel (DAPI - blue, insulin, proinsulin - green, VMAT2 - red). Scale bar represents 10 pM.

[0032] Fig. 17 shows that Beta cell vesicles containing proinsulin are larger in diameter than vesicles containing insulin. Cultures of human beta cells were fixed for immunohistochemistry with stained with anti-insulin (Left Panels) or anti-proinsulin (Right Panels) and nuclei counterstained with DAPI and imaged (Top Panels). The grey scale insulin or proinsulin images (Middle Panels) were processed by OpenCFU to obtain measurements of the particle diameters identified by the program and bounded by blue boxes (Bottom Panels). Scale bar represents 10 pM.

[0033] Fig. 18 shows that DDTBZ and BODIPY FL C5-Ceramide staining identify nacent and more mature beta cell vesicles. Cytoplasmic areas relative devoid of DDTBZ avid vesicles stain well with BODIPY FL C5-Ceramide to reveal the trans- golgi network. Vesicles still associated with the network are visible in the inset of the pseudocolor merged image (DDTBZ-blue, BODIPY FL C5-Ceramide-green).

[0034] Fig. 19 shows the OpenCFU vesicle counting in images of live beta cell cultures stained with DDTBZ and FluoZin™-3 AM zinc probe. Left panel. Merged imaged of DDTBZ channel (blue) and FluoZin™-3 AM zinc probe channel (green). Right Panel. Vesicles identified and counted by Open CFU program used in the vesicle morphometry studies in Fig. 8. 504 vesicles were identified by OpenCFU versus a manual count of 463. Scale bar represents 10 mM.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0035] One embodiment of the present disclosure is a compound (Dansyl dihydrotetrabenazine, DDTBZ) having the following structure:

[0036] Dansyl dihydrotetrabenazine (DDTBZ) exemplifies fluorescent probes that bind VMAT2, including human VMAT2, with high specificity and affinity. However, the present disclosure contemplates probes in which the VMAT2 binding moiety has a structure that varies from DDTBZ but binds to VMAT2 with high affinity. As used herein,“high affinity” means that the probe binds to VMAT2 with a K _d of 10 nM or less, preferably 1 nM or less. In the probes of the present disclosure, the VMAT2 bindng portion is associated with, e.g., covalently bonded to a detection agent. The detection agent may be any moiety that is capable of being detected (directly or indirectly) and that does not significantly interfere with the affinity and specificity of the binding moiety. In the present disclosure, fluorescent agents such as dansyl are exemplified but other detection agents may be used. Non-limiting examples of such detection agents include peroxidase enzyme, luciferase, fluorophore, fluorescent protein, fluorescent dye, lanthanide, quantum dot, biotin, digoxin, hapten, and epitope. When a fluorescent detection agent is used, it is contemplated that such agent may emit a signal across the visible light spectrum thus providing signals of many different colors. Such fluorescent agents are well known in the art and may be obtained commercially from any number of sources including BD Biosciences, PerkinElmer, Sigma Aldrich and others. [0037] Another embodiment of the present disclosure is a vesicular monoamine transporter type 2 (VMAT2) binding probe comprising a compound disclosed herein.

[0038] Another embodiment of the present disclosure is a kit comprising the probe disclosed herein together with instructions for its use.

[0039] Another embodiment of the present disclosure is a method for determining the maturity of a beta cell in the pancreas of a subject. This method comprises:

a) obtaining and culturing beta cells from the subject; b) staining the beta cell culture with DDTBZ and at least one additional fluorescent agent; c) obtaining at least one computerized image of the stained beta cell culture; and d) morphometrically analyzing the computerized image in order to determine the maturity of the beta cell.

[0040] In some aspects of this embodiment, the additional fuorescent agent is selected from the group consisting of: NeuroSensor 521 , LysoTracker ^® Red DND-99, FluoZin-3 AM, BODIPY™ FL C5-Ceramide, DAPI, and combinations thereof.

[0041] In some aspects of this embodiment, the morphometric analysis comprises determining at least one of the following parameters: cell shape, degree of agent binding, vesicle count, vesicle size, and combinations thereof.

[0042] In some aspects of this embodiment, the computerized image is obtained using fluorescence imaging (FI).

[0043] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. In some embodiments of the present disclosure, the subject is a mammal, preferably a human.

[0044] Another embodiment of the present disclosure is a method for diagnosing a metabolic disorder in a subject. This method comprises: a) obtaining beta cells from the subject and culturing with a moderate glucose concentration basal medium; b) staining the beta cell culture with DDTBZ and FluoZin-3 AM; c) obtaining at least one computerized image of the stained beta cell culture; d) morphometrically and quantitatively analyzing the computerized image to determine the average vesicle diameter of the cultured beta cells; e) exchanging at least a portion of the culture medium in step (a) with a low glucose concentration basal medium and incubating; f) repeating steps (b) to (d) with the cultured cells from step e) to determine the average vesicle diameter of the cultured beta cells; and g) if the average vesicle diameter of the beta cells determined in step (f) is not significantly reduced relative to the average vesicle diameter of the beta cells determined in step (d), it is indicative of the presence of a metabolic disorder in the subject.

[0045] In some aspects of this embodiment, the computerized image is obtained using fluorescence imaging (FI).

[0046] In some aspects of this embodiment, the moderate glucose concentration is about 10 to 12 mM. In some aspects of this embodiment, the low glucose concentration is about 2.5 mM.

[0047] In some aspects of this embodiment, the method further comprises initiating a treatment protocol for a subject determined to have a metabolic disorder based on the outcome of step (g).

[0048] In some aspects of this embodiment, the method further comprises adjusting a treatment protocol for a subject based on the outcome of step (g).

[0049] As used herein, "metabolic disorder" refers to any problem in the body that causes loss of metabolic homeostasis, and "pancreatic beta cell associated disorder" refers to any disorder or disease characterized by changes in beta cell mass or function including, but not limited to, diabetes, preclinical diabetes and hypoglycemic disorders including insulinoma. As used herein, "diabetes" refers to any disorder of glucose metabolism leading to hyperglycemia and includes both type 1 and type 2 diabetes.

[0050] In some aspects of this embodiment, the disorder is a pancreatic beta cell associated disorder. In some aspects of this embodiment, the disorder is an insulinoma. In some aspects of this embodiment, the disorder is diabetes. In some aspects of this embodiment, the disorder is type 1 diabetes. In some aspects of this embodiment, the disorder is type 2 diabetes. In some aspects of this embodiment, the disorder is preclinical type 1 diabetes.

[0051] Another embodiment of the present disclosure is a method for identifying the presence of immature beta cells in the pancreas of a subject. This method comprises:

a) administering an effective amount of DDTBZ to the subject; b) obtaining at least one computerized image of at least a portion of the pancreas of the subject; and c) quantitatively analyzing the computerized image in order to identify the presence of immature beta cells in the pancreas of the subject.

[0052] As used herein, an“immature beta cell” refers to a beta cell having a reduced threshold for glucose-stimulated-insulin-secretion (GSIS), secreting insulin in response to a lower glucose concentration than a mature beta cell.

[0053] In some aspects of this embodiment, the computerized image is obtained using fluorescence imaging (FI).

[0054] Another embodiment of the present disclosure is a compound (iodo- DDTBZ) having the following structure:

[0055] In some embodiments, the iodine is radioiodine selected from ¹²³ l, ¹²⁵ l, and ¹³¹ l. In some embodiments, the iodine is ¹³¹ l.

[0056] Another embodiment of the present disclosure is a method for determining the maturity of a beta cell in the pancreas of a subject, comprising:

e) obtaining and culturing beta cells from the subject; f) staining the beta cell culture with a radioactive compound disclosed herein; g) obtaining at least one computerized image of the stained beta cell culture; and h) morphometrically analyzing the computerized image in order to determine the maturity of the beta cell.

[0057] In some embodiments, the computerized image is obtained using Single Photon Emission Computerized Tomography (SPECT).

[0058] As used herein, "administration," "administering" and variants thereof means introducing a composition, such as a detectable agent that has a high affinity and selectivity for VMAT2, e.g., a VMAT2-specific fluorescent-ligand into a subject. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes self-administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration can be carried out by any suitable route.

[0059] As used herein, "effective amount" refers to an amount of a VMAT2- specific probe, e.g., a fluorescent-ligand effective to provide an image of the region of interest using, e.g., fluorescent imaging. The amount of the probe, e.g., fluorescent-ligand, that is effective in providing an image will vary depending the particular factors of each case, including the type of probe, e.g., fluorescent-ligand used, the subject's weight and the method of administration. These amounts can be readily determined by the skilled artisan.

[0060] As used herein, “fluorescence imaging” or “fluorescence microscopy” refers to a technique for examining fixed and living specimens, e.g., cells or organs, and both topological features as well as fluorescence intensity variations can be evaluated in a quantitative manner. Fluorescent compounds emit light in a defined spectral range when excited with light of a specific wavelength. Fluorescence imaging makes use of this phenomenon and allows for the selective and specific detection of molecules at small concentrations with a good signal-to-noise ratio.

[0061] It is also within the confines of the present disclosure that a formulation containing a VMAT2-specific fluorescent-ligand may be further associated with a pharmaceutically acceptable carrier, thereby comprising a pharmaceutical composition. Accordingly, the present disclosure further provides a pharmaceutical composition, comprising a VMAT2-specific fluorescent-ligand and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others. Formulations of the pharmaceutical composition may be conveniently presented in unit dosage.

[0062] The pharmaceutical formulations of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, the fluorescent-ligand may be brought into association with a carrier or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents and the like) also may be added. The choice of carrier will depend upon the route of administration. The pharmaceutical composition would be useful for administering the fluorescent-ligand of the present disclosure to a subject. The fluorescent-ligand would be provided in an amount that is effective to provide one or more image of a region of interest of the subject. That amount may be readily determined by the skilled artisan, as described above.

[0063] In some aspects of this and other embodiments, the subject is a mammal. Preferably, the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammal is a human.

[0064] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population may fail to respond or respond inadequately to treatment.

[0065] As used herein, the terms“ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject, preferably a human.

[0066] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0067] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

EXAMPLES

[0068] The following examples are provided to further illustrate certain aspects of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.

Example 1

Materials and Methods

Chemicals

[0069] Enantiomerically pure (+)-9-0-Desmethyl-a-Dihydrotetrabenazine was obtained from BOC Sciences, Long Island, NY and represented a convenient starting material for synthesis. All organic synthetic reagents were obtained from Sigma Millipore (St. Louis, MO). All other reagents were of the highest commercially available quality.

Synthesis of DDTBZ (Dansyldihydrotetrabenazine).

[0070] 2-hydroxy-3-isobutyl-9-methoxy-1 ,3, 4, 6, 7, 11 b-hexahydro-2H-pyrido[2, 1 - a]isoquinolin-8-yl 5-(dimethylamino)naphthalene-1 -sulfonate (SP-III-132C). (+)-9-0- Desmethyl-a-Dihydrotetrabenazine (20 mg, 0.06 mmol) was dissolved in 2.5 mL dichloromethane and a catalytic amount of 4-Dimethylaminopyridine (DMAP) was added. The reaction was cooled to 0 °C and slowly, drop-by-drop, dansyl chloride (17.5 mg, 0.06 mmol) dissolved in 2.5 mL dichloromethane was added. Reaction was stirred for 2 h at 0 °C and for 24 h at room temperature. Reaction mixture was concentrated under vacuum and purified using flash chromatography in 1 % methanol/dichloromethane solvent system. The product was obtained as a yellow- green oil.

Purification and analysis of DDTBZ probe

[0071] Column flash chromatography was carried out using E. Merck silica gel 60F (230-400 mesh) (Sigma Millipore). 1 H NMR spectra were recorded on a Varian- 400 MHz NMR spectrometer, in CDCH3 (referenced to internal Me4Si at dH O ppm). The high performance liquid chromatography (HPLC) system: a Shimadzu LC-6AD pump equipped with an SPD-M10A PDA detector was used and a single Hamilton Co. (Reno Nevada) manual injector. Semi-preparative chromatography was performed using SymmetryPrep™ C-18 C18 7 pm, 19x150mm column, (Waters). Mobile phase details: Buffer A was composed of distilled water, and buffer B 0.25% acetic acid in 90% acetonitrile. Purity was confirmed by analytical LC/MS recorded with a Shimadzu system. Elution started with water (95%, +0.1 % formic acid) and acetonitrile (5%, +0.1 % formic acid) and ended with acetonitrile (95%, 0.1 % formic acid) and water (5%, 0.1 % formic acid) and used a linear gradient at a flow rate of 0.2 mL/min.

[0072] ESI-MS spectra were obtained using a mass spectrometer Agilent ifunnel Q-TOF 6550 LC-MS with source Dual Agilent Jet Stream ESI (Dual AJS-ESI). The solution of the samples was directly injected into the ESI source using an autosampler FIA at flow rate of 1 pL min 1 at 25 °C. The solutions were eluted in a gradient of 20 % water (0.1 % formic acid) and 80 % methanol (0.1 % formic acid) for ESI(+) spectra. Mass spectra of the compounds by ESI-MS were acquired using the following operating conditions: capillary voltage ±3 kV, drying gas flow 10 L min 1 , drying gas at 250 °C, nebulizer gas at 50 psi. The system was operated for acquisitions in the mass range of 50-1500 m/z.

Characterization of UV-Visible emission and absorption profiles of DDTBZ

[0073] Absorption spectra in the 250-750 nm range were recorded on a Biochrom WPA 80-3003-75 Model Biowave II UVA/isible Life Science Spectrophotometer. Emission spectra were recorded on a Thermoscientific Lumina Fluorescence spectrometer, using a 150 W Xenon Lamp and the excitation source set to the absorbance maximum of the compound in the 300-700 nm range. The spectral bandwidth was 5 nm, with a 2 second integration time. Data were acquired with Luminous software.

Cell Culture

[0074] The cell line 293-HEK was obtained from the ATCC (Manassas, VA) and cultured according to the supplier's recommendations. HEK-DAT/mCherry-VMAT2 transfectant cells were obtained as a kind gift from Dr. G. Miller (Emory University, Atlanta, GA) and have been fully described elsewhere (Bernstein et al. , 2012). These cells were cultured at 37 °C and 5% C02 in DMEM based selection media , supplemented with 10% FBS, 1 % GlutaMax solution and 100 ug/ml Zeocin (In vivoGen, San Diego, CA and 500 ug/ml G418 ( Millipore Sigma, St. Louis, MO).

[0075] Institutional Review Board approved human islets, isolated from cadaveric nondiabetic and diabetic donors, were obtained from the Integrated Islet Distribution Program (IIDP) (City of Hope National Medical Center). The average purity and viability of the islets obtained from control donors was 89 ± 2% and 95 ± 1 % (SEM), respectively. The average age and body mass index of the donors was 47 ± 3 years and 32 ± 3 kg/m2 (SEM), respectively. Upon receipt, the isolated human islets were cultured in supplemented CMRL-1066 media for no longer than 1 day, before being subcultured as previously described (Phelps et al. , 2017, Cianciaruso et al. , 2017). Briefly, human islets were washed and dissociated in 0.05% Trypsin/EDTA solution. The dissociated islets were filtered through a sterile 40 mM nylon filter and collected and washed in minimum essential medium (MEM) with GlutaMAX (Gibco), 12 mM glucose, 5% FBS, 1 mM sodium, pyruvate, 10 mM HEPES, 100 U/ml Penicillin and 100 U/ml Streptomycin and 1x B-27 Supplement (Gibco, Gaithersburg, MD). Cells were seeded at 10,000-30,000 cells/cm2 on Collagen IV (Millipore Sigma) coated coverslip chambers (Cellvis, Mountain View, CA) in the same medium and allowed to reach full adherence (4-5 days) before live cell imaging or immunohistochemistry. Newborn porcine islets were prepared as previously described (Ramji et al., 2011 ) and processed for two dimensional culture as described above, with the exception that Hams F10 media was used for culture.

[0076] In some experiments, 293 HEK or HEK-DAT/mCherry-VMAT21 transfectant cells were seeded into Collagen IV coated coverslip chambers or sterile black 96-Well Glass Bottom Microplates (Greiner Bio-One SensoPlate) previously coated with poly-L-Lysine and cultured in their corresponding media to approximately 85% confluence or chambered coverslips. Cell were incubated in "imaging media' consisting of phenol-red free RPMI 1640. media (Caisson Labs, Smithfield, UT) supplemented with 95 mg/100 ml defatted bovine serum albumin (BSA), 1 ml/100 ml Pen/Strep 100 x solution (Corning) , 84 mg/100 mis Sodium Bicarbonate, 5 mM HEPES, and 12 or 5 mM mM Glucose) supplemented with the fluorescent probe and/or (+) DTBZ at the indicated concentrations. Cultures were incubated in the dark for 45 minutes at room temperature, washed twice with imaging media without probes, and then incubated in probe free imaging media for a further 30 minutes at 37 °C in a humidified 5% C02 atmosphere.

Fluorescent ligand binding experiments

[0077] Semi confluent 293 HEK or HEK-DAT mCherry-VMAT2 transfectant cell cultures, seeded into 96 well plates, were washed once in room temperature imaging media. Supernatants were then replaced with imaging media supplemented with DDTBZ and (+) DTBZ at the indicated concentration and further incubated as described above. Cell associated fluorescence measurements were performed on a Biotek Synergy 2 instrument, using the following parameters: DDTBZ, excitation at 320 nM with a 40 nm band pass filter, emission at 500 nm with 20 nm bandpass filter, mCherry fluorescent protein, excitation at 530 nM with a 25 nm band pass filter, emission at 590 nm with 35 nm bandpass filter. The cell bound fluorescence signal from DDTBZ was normalized to the intensity of VMAT2-mCherry 590 nm fluorescence signal which served as a surrogate measure of cell number per well. Microscopy and Live Cell Imaging

[0078] A Zeiss Axiovert 135 inverted epifluorescence microscope fitted with two independently controlled collimated light sources, 5.1 W Solis 385 nm LED or a 4.2 W Solis 400-700 nm LEDs (Thorlabs Inc., Newton, NJ) and Zeiss a 50x LD EC Epiplan-Neofluar (0.55 NA), 63 x A Plan (0.8) and 100x Plan Neofluar (1.3 NA) objectives were used for cell imaging. Specific excitation and emission wavelengths where obtained with the following filter sets; A) Excitation 330WB80, Dichroic 400DCLP, Emission 495BP30 (used for DAPI and DDTBZ), B) Excitation 470QM40, Dichroic 505 DRLP, Emission 535QM50 (used for FluoZin-3, AM or NeuroSensor 521 ), and C) Excitation 560QM55, Dichroic 595 DCLP, Emission 645QM75 (used for LysoTracker Red DND-99) (all from Omega Optical, Brattleboro, VT). DDTBZ was imaged first using the 385 nm source. The 385 nm source was then extinguished and the broadband 400-700 nm source turned on for subsequent image acquisition. In this optical system, at maximum magnification under oil, the x-y resolution was around 220 nm (given by the Abbe limit) and the depth of field of approximately 250 nm (given by the Shillaber equation). Under these culture conditions, most cells could be captured in their entirety using a single focal plane.

[0079] Images were projected onto a Lumenera Infinity 2 monochrome 2 megapixel camera (Ottawa, Ontario, CA) and acquired with Lumenera Infinity Capture or IMAGE J software. Image data was exported as single 1392 x 1040 pixel TIFF files and further processed using IMAGEJ (Schneider et al. , 2012), OpenCFU (Geissmann, 2013) and/or Adobe Photoshop CS3. The size of objects within the images captured was obtained via calibration of ImageJ software with a stage micrometer (0.1 mm in 0.002mm)(Ted Pella, Inc, Redding, CA).

[0080] Cover glass chambered cell cultures were removed from the incubator and the culture media aspirated, washed twice with imaging media. Cultures were then incubated in imaging media containing 75 mM DDTBZ for 45 min at room temperature, washed twice in imaging media and then incubated imaging media for a further 30 minutes at 37 °C in a humidified 5% C02 atmosphere. In the indicated experiments, prior to staining, beta cell cultures were preincubated in imaging media containing 12 mM glucose or 2.5 mM Glucose for 1 hr at 37 °C , with 5% C02. Coverslip chamber cultures were then moved to the microscope environmental chamber with temperature regulated heated stage and objective warmer ((CSH-1 and OW, Warner Instruments, Hamden, CT). The cultures were maintained at 37 °C in 5% C02 in this chamber. Cells were imaged at the indicated wavelengths. In some experiments, in addition to DDTBZ, cultures were also stained with 50 nM LysoTracker® Red DND-99 (Molecular Probes/Thermo Fisher Scientific, Waltham, MA), 2 mM FluoZin™-3, AM, 5 mM BODIPY™ FL C5-Ceramide complexed to BSA (both from Invitrogen/Thermo Fisher Scientific, Waltham, MA), or 100 nM NeuroSensor 521 ( Millipore Sigma). Throughout the 1 -2 hour imaging session, we observed no signs of toxicity such as cell rounding and detachment. Identical cultures treated with reserpine (10 pM), a fluorescent but irreversible VMAT2 inhibitor, showed cell rounding and detachment within 30 minutes of treatment (data not shown).

Immunohistochemistrv

[0081] Monolayers of pancreatic islet cells were washed in phosphate buffered saline (PBS) and fixed with 4% EM-grade paraformaldehyde in PFA in PBS (Electron Microscopy Sciences) at room temperature for 15 minutes. Samples were permeabilized in PBS + 0.3% Triton X-100 and blocked with PBS + 0.2% Triton X- 100 with 10% goat or rabbit serum for 60 minutes at room temperature. Primary antibodies (listed below) were incubated overnight at 4 °C. Alexa Fluor conjugated secondary antibodies (Invitrogen) were incubated in 0.3% Triton X-100 in PBS plus BSA for 60 minutes at room temperature. Coverslips were mounted with Vectasheild HardSet Antifade Mounting Medium with DAPI (Vector labs, Burlingame, CA). The following primary antibodies and dilutions were used for immunofluorescent staining: anti-insulin rabbit monoclonal antibody, 1 :1000 (Boster , #M00067-1 ), Human/Mouse Proinsulin Alexa Fluor® 488-conjugated mouse monoclonal antibody, 1 :200 (IC13361 G) and Human VMAT2 Alexa Fluor® 594-conjugated mouse monoclonal antibody, 1 :200 (FAB8327T) (both from R and D systems, Minneapolis, MN). A goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 430 conjugate, 1 :1000 (Invitrogen , #A-11064) was used to detect the rabbit primary antibody above.

Glucose stimulated Insulin secretion Assays

[0082] The human beta cell cultures (prepared from two donors 324 and 228/8 wells each) were studied on day 5 (in 12 mM Glucose) and day 7 (last two days of incubation in 4 mM Glucose). The cultures were washed 4 x with Krebs Buffer Solution (HEPES 25 mM, NaCI 115 mM, NaHCOs 24 mM, KCI 5 mM, MgCI ₂ 1 mM, CaCI ₂ , 2.5 mM, BSA 0.1 % w/v with 2.5 mM Glucose. The culture chambers were next filled with 1 ml of the warmed Krebs 2.5 mM glucose solution and the cultures were incubated (5% C0 ₂ ) for 1 hr at 37 °C degrees (similar to the protocol described for the vesicle morphometry). Next, the Krebs buffer solution was removed and replaced 1 ml Krebs buffer solution containing either 2.5 mM glucose or 16 mM Glucose and incubated again at 37 °C. Sixty minutes later the Krebs buffer solution was harvested into labelled tubes. Residual Krebs buffer was aspirated and replaced with 250 ul of warm 5% SDS. The chambers were incubated for 30 min at 37 °C and then the SDS solution was harvested into labelled tubes. Insulin assays were performed using an ALPCO human insulin ELISA following the manufactures’ recommendations. The DNA content of the well was measured using the Promega Quantitiec DNA assay following the manufactures recommendations. Aborbance and fluorescence measurements were made using the Synergy 2 multiplate reader (Biotek). The mean normalized insulin concentrations in the harvested Krebs buffer were calculated from replicate chambers and triplicate determinations of insulin concentration in the sample. Each chambers mean insulin concentration was normalized to the DNA content of the well.

Data analysis

[0083] Descriptive statistics, means, modes, standard deviations, standard errors as well as frequency distributions were calculated by Excel. Statistical significance was accepted at p values less or equal to 0.01. For vesicle morphometry, we used the open source program OpenCFU on a windows platform (Geissmann, 2013) which outputs its measurements in an Excel compatible format. Images from 20-40 cells from each culture condition and donor were acquired with the 100 x objective and resulted in diameter measurements from about 10 ⁴ vesicles from the each culture condition.

Example 2

Physiochemical characterization

[0084] The synthetic strategy for the probe (Fig. 1 ) was based on the structures of the specific VMAT2 inhibitor dihydrotetrabenazine (Fig. 2, Structure 1 ), the validated, subnanomolar Kd PET probe for VMAT2, 18F-fluoropropyl dihydrotetrabenazine (FPDTBZ) (Fig. 2, Structure 2), the radiosynthetic precursor of 18F-FPDTBZ, (+)-9- O-Desmethyl-a-Dihydrotetrabenazine (Fig. 2, Structure 3). The probe synthesized had a molecular weight of 538 g/mol by ESI-MS (m/z+1 = 539). Characterization of absorption and emission spectra of DDTBZ revealed maxima at Aex = 339 nm and Aem = 523 (Fig. 3). As expected, the excitation and emission maxima were similar to the parent fluorescent reporter dansyl chloride. Neither the precursor nor DTBZ showed significant fluorescence at 523 nm at the concentrations tested (<100 mM).

Example 3

DDTBZ colocalizes with and binds preferentially to VMAT2 positive cells

[0085] To demonstrate the specificity of DDTBZ to VMAT2, various concentrations of DDTBZ was added to live cultures of untransfected FIEK 293 cells and FIEK 293 cells transfected with the VMAT2-m Cherry fusion protein. Cells were then imaged for DDTBZ, followed by VMAT2-m Cherry at the indicated wavelength (Fig. 4). We found DDTBZ bound preferentially to VMAT2 positive cells and that there was significant overlap of the DDTBZ signal and mCherry VMAT2 signal.

[0086] We found (+) DDTBZ signal significantly overlapped with mCherry VMAT2 signal. Next, we extended the studies of (+) DDTBZ binding to three other non-beta cell lines to characterize the possible specific and non-specific binding of the probe. FIEK 293 cells, the untransfected counterparts of FIEK-DAT mCherry-VMAT2 cells studied above, PANC1 cells, derived from a human pancreatic carcinoma of ductal cell origin and 5637 cells, derived from a human uroepithelial carcinoma were seeded into coverglass chambers slides and allowed to attach and proliferate. FIEK 293, PANC1 and 5637 cells were incubated with (+) DDTBZ, LysoTracker Red and Nuclear Mask Deep Red and the cells imaged at the indicated wavelengths (Fig. 5). The collection of DDTBZ fluorescent signal was performed under a higher gain setting relative to that used for imaging, LysoTracker Red and Nuclear Mask Deep Red signals, to allow visualization of possibly weak signals. We found that (+) DDTBZ fluorescence in FIEK 293 cells was greatest in the nuclear region and perinuclear regions of the cells, but not found in vesicles. LysoTracker red staining of FIEL 293 cells show few vesicles. Similarly (+) DDTBZ fluorescence in PANC-1 cells was associated with the nucleous and some uncharacterized cytoplasmic structures. LysoTracker red staining of PANC-1 cells show many more small vesicles than FIEK 293, but (+) DDTBZ fluorescence was not associated with these vesicles. The 5637 cell line showed abundant vesicular staining with (+) DDTBZ, as well as perinuclear and nuclear staining. LysoTracker red staining of 5637 cells show many more small vesicles than PANC-1 cells. (+) DDTBZ vesicular fluorescence was associated with a partially overlapping set of Lysotracker red avid vesicles, but some vesicles stained only with (+) DDTBZ. Using the greyscale values from the cell images, the vesicular staining in 5637 cells was 2-5 times the cytoplasmic background depending on the location of the vesicles. Examination of the public gene expression databases revealed that HEK 293 cell do not express SLC18A2 transcripts (a.k.a VMAT2) (Lugowski et al. 2018), while PANC-1 cells express SLC18A2 transcripts weakly (i.e. in the bottom 0.3% of the >40 k different genes probed) (Thu et al. 2014). In a previous report, 5637 cells were used to deposit an extracellular matrix on cover slips suitable for human beta cell culture (Phelps et al. 2017). 5637 cells have also been used a source of secreted colony stimulating factors (Platzer et al. 1985), but were not recognized for expression of VMAT2. The vesicular staining by (+) DDTBZ correctly identified 5637 cells as expressing SLC18A2 transcripts (Lee et al. 2017).

[0087] To better quantitate the specificity, displaceability and saturability of DDTBZ binding to cellular VMAT2, we repeated the binding experiments in 96 well plates suitable for fluorescent imaging of HEK-DAT mCherry-VMAT2 and untransfected HEK 293 cells. Cell cultures were incubated in DDTBZ and/or DTBZ, washed and the bound fluorescence quantitated (Fig. 6). Binding of DDTBZ was saturable, and displaceable by increasing concentrations of DTBZ in HEK -DAT, mCherry-VMAT2 transfected cells. The untransfected HEK 293 cells bound >50 times less DDTBZ relative to the VMAT2 transfected cells.

Example 4

VMAT2 and dopamine colocalize in the vesicles of cultured beta cells

[0088] We previously demonstrated that beta cells are the primary storage depot for dopamine in the pancreas (Veluthakal and Harris, 2010), insulin and VMAT2 immunoreactivity colocalize almost exclusively in the beta cells of human islets (Saisho et al., 2008; Freeby et al., 2012), and human islets release both insulin and dopamine in response to glucose challenge (Simpson et al., 2012). We took advantage of the two-dimensional human beta cell culture system described by Phelps et al (Phelps et al., 2017) to examine the expression of VMAT2 in dissociated human and porcine islet cell cultures. The DDTBZ excitation spectra peaks in the near UV region of the spectrum and allowed us to image other fluorescent probes in the same cultures. NeuroSensor 521 is a fluorescent turn-on sensor that allows for the selective recognition and sensing of norepinephrine and dopamine in live cells (Hettie et al. , 2013). Since islet beta cells do not express Dopamine-Beta- Hydroxylase responsible for the transformation of dopamine into norepinephrine (Adeghate and Donath, 1991 ) and analysis of human islet culture supernatants by HPLC coupled with electrochemical detection demonstrate dopamine but fail to show significant amounts of norepinephrine (data not shown), we concluded that NeuroSensor 521 would enable us to visualize the presence of vesicular dopamine (DA) in these cultures. Beta cell cultures were also stained with LysoTracker® Red DND-99, a fluorescent probe specific for acidic organelles such as lysosomes as well as the insulin containing beta cells vesicles which are reported to have resting pH around 5.9-6.2 (Tompkins et al., 2002). In Fig. 7 representative images of cells stained with DDTBZ, NeuroSensor 521 and LysoTracker Red DND-99 are presented. Islet cell cultures treated with 20 mM DDTBZ and 5 fold molar excess of DTBZ showed a 50-100 fold loss in fluorescent intensity (data not shown).

[0089] Merged pseudocolor images of DDTBZ, NeuroSensor 521 and LysoTracker® Red DND-99 stained cultures revealed a morphologically heterogenous population of cells with respect to cell shape, degree of probe binding and vesicle size. DDTBZ , NeuroSensor 521 , Lysotracker red binding represented largely overlapping sets, but many vesicles showed strong binding of only one or two of the fluorescent probes. Not all cells bound DDTBZ and NeuroSensor 521 and are likely to represent the non-beta cell constituents of the islet cultures (see Fig. 12). Similar results were found in cultures of dispersed porcine islet cells treated with DDTBZ, NeuroSensor 521 and LysoTracker® Red DND-99 (Fig. 15).

[0090] Murine islets have been reported to express lower amounts of VMAT2 relative to its expression in human islets24 . We examined the binding pattern of (+) DTBBZ in cultures of bTO-6, an insulin secreting mouse cell line derived from transgenic B6D2 mice harboring the SV40 T-antigen driven by the upstream enhancer-promoter region of the rat insulin II gene25 . We found that (+) DDTBZ staining (Fig. 13) was heterogenous as we could identify cells clusters devoid of (+) DDTBZ positive cells (Fig. 13, upper panel), cell clusters containing both (+) DDTBZ positive and negative cells (Fig. 13, lower panel) and clusters composed of almost entirely of (+) DDTBZ positive cells. Within the group of bTO-6 cells and cell clusters staining positively with (+) DDTBZ, the number of (+) DDTBZ positive vesicles per cell (mean = 2.2 versus 208) as well as the vesicle diameters (mean = 512 nm versus 699 nm) were significantly (p < 0.05, two tailed test) smaller than vesicles imaged in (+) DDTBZ positive human islet cells.

[0091] To demonstrate that beta cell vesicular binding of (+) DDTBZ was displacable, islet cell cultures treated with 30 mM (+) DDTBZ and an 8 fold molar excess of racemic (±) DTBZ (corresponding to a 4 fold molar excess of (+) DTBZ). Vesicles in these cultures showed an average 50% reduction in average vesicular fluorescent intensity (Fig. 14). Next, we examined cultures of dispersed porcine islet cells treated with (+) DDTBZ, NeuroSensor 521 and LysoTracker® Red DND-99 (Fig. 15). Similar results to human islets cultures were obtained.

Example 5

VMAT2 and Zinc colocalize in the vesicles of cultured beta cells

[0092] The role of zinc in the formation of mature insulin secretory vesicles has been reviewed by Li (Li, 2014). Briefly, translated preproinsulin in the lumen of the rough ER is cleaved to form proinsulin. Proinsulin is transported to the trans-Golgi network for packaging into secretory granules where it is converted to insulin by selective cleavage of the C-peptide. Insulin is initially found in its monomeric form, but as it accumulates, a dimeric conformation is favored. Intracellular Zn ²⁺ is transported via ZnT8 into the vesicles where it facilitates the formation of a 2-Zn ²⁺ - trimer of dimers (i.e. hexameric insulin) in a process that has been reported to be stabilized by the presence of dopamine (Palivec et al. , 2017). On this basis, we have used intravesicular Zn ⁺² combined with the presence of VMAT2 as a surrogate marker for insulin vesicles.

[0093] Fluorescent zinc probes have been used to image intracellular Zn ²⁺ in beta cells (Zhou et al., 2010, Jayaraman, 2008) but not at the resolution levels used in current experiments. We selected FluoZin-3, AM, an on-off selective fluorescent Zn2+ probe (Gee et al., 2002). The probe is prepared as an acetoxymethy ester which enhances cell permeability, yet de-esterifies intracellularly as an aid to maintenance of the signal. Merged pseudocolor images of DDTBZ, FluoZin-3, AM and LysoTracker® Red DND-99 stained cultures (Fig. 8) revealed a morphologically heterogenous population of cells with respect to cell shape, degree of probe binding and vesicle size. DDTBZ, FluoZin-3, and LysoTracker Red triple positive vesicles could be identified in a majority of cells. In addition, we could identify at least three subsets of triple positive cells; 1 ) Cells containing mostly larger vesicles (diameters 600-2000 nm) (Fig. 8, row 1 ), mixed large and small vesicles (Fig. 8, row 2), and cells containing mostly smaller vesicles (diameters < 480 nm) (Fig. 8, row 3). Some cells showed DDTBZ positive vesicles, yet only a few vesicles binding FluoZin-3 and a weak cytoplasmic signal in the green channel (Fig. 8, row 4).

[0094] Lastly, common features of the vesicles visualized by DDTBZ (Fig. 7, Fig. 8 and Fig. 18), were the occasional apparent entrainment of vesicles within the beta cell cytoplasm (Fig. 8, rows 1 and 6), a wide range of vesicle diameters, and perinuclear regions relatively void of vesicles staining with DDTBZ (Fig. 8, row 1 and 2, Fig. 7, row 2) compared to adjacent areas. We speculated that the larger vesicles might be enriched for proinsulin polypeptide and the smaller vesicles enriched with the mature processed form insulin. Human beta cells cultures were fixed and processed for immunohistochemistry with anti-VMAT2 antibodies and anti-proinsulin or anti-insulin antibodies. Confirming our previous findings using formaldehyde fixed paraffin embedded whole pancreas tissue (Saisho et al. , 2008), we found that VMAT2 and insulin immunoreactivity colocalized in the cytoplasm of fixed beta cells (Fig. 16). Additionally we found that larger VMAT2 positive vesicles were indeed enriched for proinsulin polypeptide (Fig. 17).

[0095] To better understand the relationship between DDTBZ binding and the morphology of human beta cells in this culture system, we stained the beta cells with DDTBZ and substituted FluoZin-3 AM with BODIPY™ FL C5-Ceramide complexed to BSA (Fig. 18). BODIPY™ FL C5-Ceramide is a well characterized stain for the trans-golgi network (Pagano et al., 1991 ). We observed that the cytoplasmic voids of DDTBZ positive vesicles represented areas containing the trans-golgi network as revealed by the binding of BODIPY™ FL C5-Ceramide. Presumably nascent secretory vesicles, as judged by their intimated association with the golgi membranes, were visible (Fig. 18, inset). Of these nascent vesicles, only a minority retained DDTBZ or LysoTracker Red (data not shown).

Example 6

Incubation of cultured beta cells in low glucose is accompanied by a reduction in average insulin vesicle size [0096] In vitro, islets, beta cells, and beta cell lines are often cultured under growth/survival permissive moderate glucose concentrations (10-12 mM Glucose). For measurements of insulin secretion from these cultures, however, the normal culture media is often exchanged with a lower glucose concentration basal medium that does not trigger insulin secretion and the cultures are allowed to "rest" for various intervals, presumably to allow the accumulation of fully processed mature insulin secretory vesicles. Once stimulated, the amplitudes of secretory responses are greater relative to cultures that have not been preincubated in low glucose basal medium. We tested the insulin secretion characteristics of the two-dimensional dispersed human b-cell culture system. The b-cell cultures were incubated normally in 12 mM glucose culture medium for five days and then tested for glucose stimulated insulin secretion (GSIS), or cultured for an additional two in culture medium containing 4 mM Glucose. Cultured b-cells were washed and then rested for 60 minutes at 37 °C in a 2.5 mM glucose buffer. This media was replaced with 1 ml of either 2.5 or 16 mM glucose buffer and incubated again for 60 minutes at 37 °C. The insulin concentration in each solution was measured by enzyme linked immunoassay and then normalized to the DNA content of the well. We next calculated a stimulation index comparing the insulin secreted at 16 mM Glucose to the insulin secreted at 2.5 mM Glucose (Fig. 9, Fig. 10 and Fig. 11 ).

[0097] There was a statistically significant increase (p < 0.05 paired t test) in the insulin secretion rate when 2.5 mM (basal) and 16 mM glucose (stimulated) were compared. The stimulation indices for all conditions were about 1.3 ± 0.2. The insulin secretion rates were less on day 7 (following 48 hr incubation in culture media supplemented with 4 mM glucose) relative to day 7.

[0098] To demonstrate the utility of identifying and quantifying VMAT2 positive, zinc positive vesicles in living beta cell cultures, we prepared beta cell cultures from two nondiabetic control donors and one donor diagnosed with type 2 diabetes mellitus (T2D). Cell cultures were either maintained in high glucose (12 mM) media or incubated in low glucose (2.5 mm) media for one-hour prior to staining with DDTBZ and FluoZin-3. Next, the cell cultures were imaged, the DDTBZ and FluoZim-3 channels combined and the diameters of DDTBZ and FluoZin-3 double positive beta cell vesicles quantified by the OpenCFU software (Fig. 19). During the one hour 2.5 mM low glucose incubation period, it is estimated that in control beta cells, the average vesicle diameter is significantly reduced by about 100 nm (two- tailed t-test, p < 0.0001 ), relative to the diameters measured under 2.5 mM glucose culture conditions (mean vesicular diameter = 734 nm) (Fig. 10, Panels A and B; Fig. 11 , Left Panel). Vesicle diameters were not reduced by 2.5 mM glucose culture in beta cells obtained from the donor diagnosed with T2D, rather an increase in mean vesicular diameter, from 688 nm to 724 nm (two-tailed t-test, p < 0.0001 ) was observed (Fig. 10, Panel C; Fig. 11 , Right Panel). When vesicular diameters were analyzed on per cell basis (Fig. 10, Panel D) from a 12 mM glucose culture, a biomodal distribution, with maxima at 300 nm and 780 nM, was observed.

Example 7

Discussion

[0099] We report on the synthesis, physiochemical and biological characterization of a fluorescent ligand for VMAT2 and demonstrate its utility in an application relevant to beta cell biology. DDTBZ differs from previous optical tracers used in CNS neurotransmission studies. These tracers, namely fluorescent false neurotransmitters (Pereira et al. , 2016, Gubernator et al. , 2009) and dansyl dopamine (May, 2013), are nonselective VMAT1 and VMAT2 substrates. Future studies should include a side by side comparison of these optical tracers in both the beta cell and neuronal cell cultures to understand their similarities and differences.

[0100] The selected application to showcase the utility of DDTBZ was a morphometric study of insulin secretory vesicles in cultured beta cells. Our principle findings are summarized as follows: 1 ) DDTBZ and dopamine staining reveal a morphologically heterogeneous population, in terms cell size, shape, vesicle number, size and contents, 2) VMAT2 and Zn ⁺² colocalize in wide range of beta cell acidic vesicle sizes, 3)The larger beta cell vesicles are enriched for proinsulin, whereas the smaller vesicles predominantly contain the processed mature insulin and both these vesicles types, immature and mature, colocalize with VMAT2, 4) In beta cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles (around 300 nM) at the expense of the larger immature insulin secretory vesicle population. Low glucose induced vesicle maturation was not observed in the beta cell cultures obtained from the donor with T2D.

[0101] Transmission and scanning electron microscopy of beta cells in situ reveals about 9-13,000 secretory vesicles per beta cell with mature dense core vesicles bearing an average diameter of 300 nm (Dean, 1973, In’t Veld and Marichal, 2010, Olofsson et al. , 2002). In these studies, immature vesicles, staining less avidly with uranylacetate (a.k.a. grey granules) have been reported but never studied morphophometrically as is detailed in this report. In this culture system, we find that only 20-40 percent of the vesicles within the beta cell population are found in the diameter range of mature granules (<390 nm). A second important difference between our results and previous findings is the number of vesicles. We have counted (either manually or by computer assisted image analysis) no more than 1 ,000 vesicles per cell staining for DDTBZ and FluorZin-3. The simplest explanation for this difference is that our lower vesicles counts are due to the smaller cell volumes encountered in this "flattened beta cell" culture model. In situ beta cells are estimated to have a 10 pm diameter (Zimny and Blackard, 1975) corresponding to a spherical volume of 525 pm ³ . The beta cells characterized in this system are better approximated by prolate ellipsoids with x, y and z axes of approximately 7, 10 and 0.25 pm , respectively, corresponding to volume approximately one-tenth that of in situ beta cells. Hence our lower vesicles counts are concordant with previous reports of vesicle counts are normalized to cell volume. The flattened and dissociated b-cells studied in this system displayed GSIS stimulation indices (S.l.) that where low (S.l. « 1.3) relative to freshly isolated islets, yet equivalent to reports of islets that have been cultured for five days (Murray et al. 2005).

[0102] In vitro human beta single cell imaging with DDTBZ reveals significant heterogeneity. Single beta cell heterogeneity has been previously documented for variety of outcome measures using different experimental systems including flow cytometry, these findings have been recently reviewed by Nasteka and Hodson (Nasteska and Hodson, 2018). Most recently VMAT2 has been shown to be a marker of immature beta cells in cultures of induced pluripotent stem cells and its specific inhibitor, DTBZ, been shown to be important regulators of beta cell maturation and beta cell mass (Sakano et al., 2016, Sakano et al., 2014, Maffei et al., 2015). The ability to probe heterogeneous populations and identify immature beta cells by DDTBZ staining may be a useful application of this probe.

Example 8

Synthesis of iodo-DDTBZ

[0103] As shown in Scheme 1 below, a bromine was introduced into aromatic part of dansyl chloride, followed by displacement of bromine with tributylstannyl group. Displacement of aromatic tributylstannyl group with iodine yielded iodinated dansyl chloride, which was consequently coupled to Desmethyl dihydrotetrabenazine (DDTBZ). In the preparation of a probe suitable for SPECT (Single Photon Emission Computerized Tomography) imaging, radioiodine ( ¹²³ l, ¹²⁵ l, ¹³¹ 1 ) was substituted for naturally abundant and non radioactive ¹²⁶ 1.

Scheme 1 . Synthesis of iodo-DDTBZ.

DOCUMENTS CITED

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3. CIANCIARUSO, C., PHELPS, E. & BAEKKESKOV, S. 2017. Preparation of monolayer cultures of primary rat and human islet cells on glass for super- resolution and live cell imaging.

4. DEAN, P. M. 1973. Ultrastructural morphometry of the pancreatic -cell.

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[0104] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety as if recited in full herein.

[0105] The disclosure being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the disclosure and all such modifications are intended to be included within the scope of the following claims.