The primary target of Melanin probe-1 is melanin, a biopolymer produced by melanocytes that is responsible for skin, hair, and eye pigmentation. Melanin is abundantly present in malignant melanoma cells, making it an excellent biomarker for imaging. The probe is based on an 18F-picolinamide structure that has a high affinity for the melanin polymer. The binding likely involves pi-pi stacking interactions between the aromatic rings of the probe and the stacked indole-5,6-quinone units of eumelanin, as well as hydrogen bonding with the carboxyl and hydroxyl groups. The 18F isotope is a positron emitter used for PET imaging, which provides high sensitivity and quantitative, three-dimensional imaging of the probe's distribution in the body. By binding to melanin, Melanin probe-1 allows for the non-invasive visualization of melanin-containing tumors. This probe is selective for melanin and does not significantly bind to other tissues, resulting in high tumor-to-background contrast ratios.

In vitro studies of Melanin probe-1 are primarily focused on validating its melanin binding specificity and cellular uptake in melanoma cells. The binding affinity of the 19F (non-radioactive) or 18F-labeled probe to melanin can be assessed using a binding assay with synthetic melanin or melanin isolated from melanoma cells. In a typical assay, various concentrations of the probe are incubated with melanin (e.g., 100 ug/mL) in PBS for 30-60 minutes at 37degC. Unbound probe is separated by centrifugation, and the amount of probe bound to melanin is determined by measuring the radioactivity (for 18F) or by HPLC (for 19F). The binding affinity (Kd) and capacity (Bmax) are calculated. In cellular uptake studies, human melanoma cell lines (e.g., B16F10 murine melanoma, A375 human melanoma, SK-MEL-28) are incubated with [18F]Melanin probe-1 (e.g., 3.7 MBq/mL) for 15-120 minutes at 37degC. Cells are washed to remove unbound probe, and the cell-associated radioactivity is measured in a gamma counter. The uptake is expressed as % of added dose per million cells. Specificity is confirmed by competitive inhibition with excess non-radioactive probe (e.g., 10-100 uM) or with a melanin-binding compound (e.g., chloroquine). Melanin probe-1 shows high uptake in melanin-producing melanoma cells but low uptake in non-melanoma cells (e.g., HEK293, HeLa) or amelanotic melanoma cells. The intracellular localization can be visualized by autoradiography or by confocal microscopy using a fluorescent analog of the probe. In vitro stability is assessed by incubating [18F]Melanin probe-1 in mouse or human serum at 37degC for 1-4 hours, followed by radio-HPLC to check for degradation. The probe exhibits good in vitro stability, with >90% intact after 2 hours.

Tumor targeting effectiveness is high for melanin probe-1 (3.7 MBq; tail vein)[1]. High in vivo stability and advantageous pharmacokinetic characteristics, such as quick excretion from the urinary system and nearly background levels of absorption for all major organs at two hours, are demonstrated by melanin probe-1. Melanin probe-1 PET has a strong potential to improve melanoma diagnosis because to its high selectivity and specificity, as demonstrated by high tumor-to-non-tumor ratios[1].

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In vivo studies in mouse models of melanoma demonstrate the excellent tumor-targeting capabilities of Melanin probe-1. In C57BL/6 mice bearing subcutaneous B16F10 murine melanomas (a pigmented model), intravenous (i.v.) administration of [18F]Melanin probe-1 (3.7 MBq, tail vein) results in rapid tumor uptake and high tumor-to-background contrast. At 0.5, 1, and 2 hours post-injection, the tumor uptake values are 12.74 +/- 1.70, 16.61 +/- 2.60, and 16.87 +/- 1.23 %ID/g (percentage of injected dose per gram of tissue), respectively. The tumor-to-muscle ratios are high (e.g., >10 at 2 hours), demonstrating excellent imaging contrasts. The probe exhibits high in vivo stability and favorable pharmacokinetic properties, such as fast clearance from the urinary system and almost background level of uptakes for all major organs at 2 hours. The high selectivity and specificity of the probe, as evidenced by high tumor-to-non-tumor ratios, highlight the potential of Melanin probe-1 PET to improve melanoma detection. The probe also shows uptake in pigmented melanoma metastases in the lungs and liver. In A375 human melanoma xenografts (less pigmented or amelanotic), the uptake is lower, but still detectable. The probe is also evaluated for imaging melanoma in other models, such as orthotopic or metastatic models. The biodistribution is assessed by dissecting major organs (heart, liver, spleen, lung, kidney, muscle, bone, brain, tumor) at various time points post-injection and measuring the radioactivity in a gamma counter. The probe is cleared rapidly via the renal route, with low retention in the liver and other non-target tissues. The excellent pharmacokinetics and low background make it suitable for clinical translation.

Non-cell-based assays for Melanin probe-1 are primarily analytical and binding studies. To measure binding to melanin, a radioligand binding assay using 18F-labeled Melanin probe-1 and synthetic melanin is performed. In a 1.5 mL Eppendorf tube, synthetic melanin (0.1-5 mg) is suspended in 0.5 mL of PBS (pH 7.4). A fixed amount of [18F]Melanin probe-1 (e.g., 100,000 cpm) is added, and the mixture is vortexed and incubated at 37degC for 30-60 minutes with shaking. Tubes are then centrifuged at 15,000 rpm for 10 minutes. The supernatant is removed, and the pellet is washed twice with 0.5 mL of PBS. The radioactivity in the pellet and the combined supernatants is measured in a gamma counter. The percentage of bound probe is calculated (pellet cpm / total cpm × 100). Non-specific binding is determined in the presence of excess non-radioactive Melanin probe-1 (e.g., 10-100 uM). The binding affinity (Kd) can be determined by a saturation binding assay, where a constant amount of melanin (e.g., 0.5 mg) is incubated with increasing concentrations of 18F-labeled probe (0.1-100 nM). Bound and free are separated as above, and the binding isotherm is fitted to a one-site binding model. For a Scatchard analysis, bound/free vs. bound is plotted. For selectivity studies, the probe is incubated with other biopolymers (e.g., cellulose, starch, protein) to assess non-specific binding. The probe should show low binding to these materials. The melting point, LogP, and other physicochemical properties of the non-radioactive precursor can be measured. The LogP (octanol/water partition coefficient) can be determined by the shake-flask method. A small amount of the 18F-labeled probe is added to a mixture of 1-octanol and PBS (1:1, 2 mL each). The mixture is vortexed for 1 min and centrifuged at 2,000 rpm for 5 min. Aliquots (100 uL) from each phase are counted in a gamma counter. The LogP is calculated as log10(cpm in octanol / cpm in water). A LogP of around 1-2 is typical for this class of probes, indicating moderate lipophilicity. For plasma stability, the 18F-labeled probe is incubated in mouse or human plasma (0.5 mL) at 37degC for up to 4 hours. At various time points (0, 30, 60, 120, 240 min), an aliquot (50 uL) is withdrawn, and the proteins are precipitated with acetonitrile (200 uL). After centrifugation, the supernatant is analyzed by radio-HPLC (C18 column, mobile phase: water/acetonitrile with 0.1% TFA, gradient). The percentage of intact probe is calculated from the area under the peak (AUC). The half-life in plasma is determined. For in vitro metabolism studies, the probe is incubated with mouse or human liver microsomes (0.5 mg/mL) and an NADPH-regenerating system for 1 hour, and the metabolites are analyzed by radio-HPLC.

Cell-based assays for Melanin probe-1 involve uptake and efflux studies in melanoma cells. Human melanoma cell lines (e.g., SK-MEL-28, A375, B16F10) are cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For uptake studies, cells are seeded in 24-well plates (2×10^5 cells/well) and allowed to attach overnight. The medium is replaced with fresh medium (0.5 mL) containing [18F]Melanin probe-1 (e.g., 37 kBq/mL). After incubation for 15, 30, 60, 120 minutes at 37degC, the medium is removed, and cells are washed three times with ice-cold PBS. Cells are lysed with 0.5 mL of 0.1 N NaOH, and the lysate is transferred to a tube for counting in a gamma counter. The protein content of the lysate is determined by BCA assay. The uptake is expressed as % of added dose per mg protein, or as cpm per 10^6 cells. To study the effect of melanin content on uptake, cells can be treated with melanogenesis enhancers (e.g., alpha-MSH, 100 nM) or inhibitors (e.g., phenylthiourea, PTU) for 48-72 hours before the uptake experiment. To study the mechanism of uptake, the experiment is performed at 4degC (to block active transport) or in the presence of endocytosis inhibitors (e.g., chlorpromazine, dynasore, or cytochalasin D). The probe is likely taken up by passive diffusion, not by endocytosis, due to its small size and moderate lipophilicity. For efflux studies, cells are loaded with the probe for 30 minutes, washed, and then incubated in probe-free medium for 0, 15, 30, 60 minutes. The amount of radioactivity remaining in the cells is measured. To assess cytotoxicity of the non-radioactive precursor, cells are treated with Melanin probe-1 precursor (0.1-1000 uM) for 24-72 hours, and viability is measured by MTT or CellTiter-Glo. The precursor is expected to have low toxicity (IC50 > 100 uM). For competitive inhibition studies, unlabeled Melanin probe-1 precursor is added to the incubation medium at various concentrations (0.1-100 uM) along with the 18F-labeled probe to determine the IC50 for binding. For specificity assessment, the uptake of the probe is compared in pigmented (B16F10, SK-MEL-28) vs. non-pigmented (A375, HEK293) cells. The uptake should be significantly higher (e.g., 5-10 fold) in pigmented cells. The probe can also be used in fluorescence-based assays if a fluorescent analog (e.g., FITC-labeled or Cy5-labeled) is available. The intracellular distribution of the probe (which is expected to colocalize with melanosomes) can be visualized by confocal microscopy after incubating live cells with a fluorescent analog. The fluorescent probe may be trapped in melanosomes due to the acidic pH (protonation) or high binding affinity.

Animal/Disease Models: C57BL/6 mice bearing B16F10 murine melanomas[1]
Doses: 3.7 MBq (100 μCi)
Route of Administration: Tail vein
Experimental Results: The tumor uptakes of Melanin probe-1 were 12.74 ± 1.70, 16.61 ± 2.60, and 16.87 ± 1.23 %ID /g at 0.5, 1, and 2 h, respectively.

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In vivo protocols for Melanin probe-1 are focused on PET imaging studies. Female C57BL/6 mice (6-8 weeks old) are injected subcutaneously with B16F10 murine melanoma cells (1-5×10^6 cells in 100 uL PBS) in the flank. When tumors reach a volume of 200-500 mm3 (approximately 10-14 days post-inoculation), the mice are used for PET imaging. [18F]Melanin probe-1 is administered via the tail vein (3.7-7.4 MBq, 100-200 uL of saline). Mice are anesthetized with isoflurane (1-2% in oxygen) and placed in a small-animal PET scanner (e.g., Inveon, Siemens). Static PET scans are performed at 0.5, 1, and 2 hours post-injection (p.i.), typically for 10-30 minutes each. For dynamic PET imaging, data is acquired continuously for 0-60 minutes p.i. After the final scan, mice are euthanized, and the tumors and major organs (heart, liver, spleen, lungs, kidneys, muscle, bone, brain) are excised, weighed, and counted in a gamma counter to measure the %ID/g and to corroborate the PET signal. Region-of-interest (ROI) analysis is performed on the PET images to obtain standardized uptake values (SUVs) for the tumor and normal tissues. The tumor-to-muscle ratio and tumor-to-blood ratio are calculated. For blocking studies, excess non-radioactive Melanin probe-1 (e.g., 10 mg/kg) is co-injected with the radiotracer to demonstrate specificity (reduced tumor uptake). For metabolic stability assessment, blood samples are collected at 5, 15, 30, 60 minutes p.i., and plasma is analyzed by radio-HPLC as described above. For autoradiography, tumor sections (10 um thick) are exposed to a phosphor imaging plate for several hours, and the plate is scanned to visualize the distribution of the probe within the tumor. The autoradiography signal should colocalize with the melanin-rich areas (identified by Fontana-Masson staining or other melanin stains). For studies in metastatic melanoma models, B16F10 cells are injected intravenously (e.g., 1×10^5 cells in 100 uL PBS) to generate lung metastases. After 14 days, the probe is injected, and the mice are imaged. The probe should accumulate in metastatic lesions in the lungs, which can be confirmed by ex vivo imaging and histology. For studies in human melanoma xenografts (e.g., A375, SK-MEL-28), female athymic nude mice are used instead of C57BL/6 mice. The imaging protocol is similar. For evaluating the probe's utility in detecting small tumors, mice with small tumors (e.g., 50-100 mm3) are used. The probe is expected to show high sensitivity due to the high target (melanin) concentration. The pharmacokinetic parameters (Cmax, Tmax, AUC, t1/2, CL) can be derived from the dynamic PET images or from blood sampling studies. For blood sampling, 10 uL of blood is collected from the tail vein at various time points (2, 5, 10, 15, 30, 45, 60, 90, 120 minutes) after probe injection. Radioactivity in each blood sample is measured in a gamma counter and expressed as %ID/g.

Pharmacokinetic data for [18F]Melanin probe-1 in mice shows that the probe has fast clearance from the urinary system and almost background level of uptakes for all major organs at 2 hours. After i.v. injection (3.7 MBq), the probe is rapidly cleared from the blood. The half-life in blood is on the order of 5-15 minutes. The probe is primarily excreted via the kidneys into the urine, with minimal retention in the liver or other organs. The biodistribution shows low uptake in non-target tissues (e.g., muscle, heart, lungs, brain) at 2 hours post-injection. The probe is stable in vivo, with minimal metabolism observed in plasma. The excellent pharmacokinetic properties contribute to the high tumor-to-background contrast and make the probe suitable for clinical translation. The non-radioactive precursor is used as a standard for calibration and for manufacturing the radiotracer. The precursor is soluble in DMSO and should be stored at -20degC, protected from light.

The 18F-labeled probe is radioactive, and its administration in animals involves ionizing radiation, which carries a risk of acute and long-term toxicity. However, the administered doses in small animals (3.7-7.4 MBq) are generally within the safe range for acute radiation effects, and no overt toxicity (e.g., weight loss, behavioral changes, death) is observed. The non-radioactive precursor is expected to have low toxicity. In cell viability assays, the precursor at concentrations up to 100 uM shows no significant cytotoxicity. Standard safety precautions for handling radioactive materials must be followed (shielding, dosimetry, contamination monitoring). The compound is for research use only and is not for human use.

[1]. Liu H, et al. Development of 18F-labeled picolinamide probes for PET imaging of malignant melanoma. J Med Chem. 2013;56(3):895‐901.

PET imaging agent

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Melanin probe-1 is an 18F-labeled PET imaging agent for the detection of malignant melanoma. The chemical name of the precursor is N-(2-(diethylamino)ethyl)-5-fluoropicolinamide. It is a small molecule (MW 239.29) that targets melanin with high affinity and selectivity. The radiolabeling is typically performed by nucleophilic substitution of a suitable leaving group (e.g., nitro or trimethylammonium) with 18F-fluoride, followed by deprotection if necessary. The radiotracer has been evaluated in preclinical models and shows promise for clinical translation. As of the search date, this probe may not be FDA-approved for clinical use, but it is available for research purposes. The non-radioactive probe is stored as a solid (or oil) at -20degC, protected from light. The radioactive form must be used within a few hours of synthesis due to the short half-life of 18F (110 minutes). This probe is a valuable tool for studying melanoma biology, evaluating the efficacy of melanoma therapies, and for the early detection of melanoma and its metastases.

C12H18FN3O

239.289226055145

239.143

1420844-62-7

71562765

Light yellow to yellow oil

1.3

1

4

6

17

234

0

CCN(CC)CCNC(=O)C1=NC=C(C=C1)F

TWRNBUQZDSVURX-UHFFFAOYSA-N

InChI=1S/C12H18FN3O/c1-3-16(4-2)8-7-14-12(17)11-6-5-10(13)9-15-11/h5-6,9H,3-4,7-8H2,1-2H3,(H,14,17)

N-[2-(diethylamino)ethyl]-5-fluoropyridine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 250 mg/mL (1044.76 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (26.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 6.25 mg/mL (26.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)