RDC Linker for Radionuclide-Drug Conjugates

Mice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3 % ID/g vs. 22.8 ± 3.8 % ID/g at 24 h) and increased tumor uptake at 120 h (13.8 ± 8.0 % ID/g vs. 6.0 ± 1.2 % ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5). Conclusions: The combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice [1].

Cell culture:[1]
The pancreatic cancer cell lines, BxPC-3 and MiaPaCa-2, were maintained at 37°C and 5% CO2 atmosphere. BxPC-3 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium carbonate, and 10% FBS. MiaPaCa-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 2.5% horse serum.

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In vitro assessment of [89Zr]Zr-Ab-AuNP:[1]
Six-well plates were seeded with 1x106 cells in 1 mL of media and allowed to adhere overnight in an incubator at 37°C and 5% CO2. The following day 1 uCi of [89Zr]Zr-5B1-AuNP was added to each well and returned to the incubator for 1, 4, 12, and 24 h. At each time point the media was collected and wells were washed with phosphate-buffered saline twice. The surface-bound fraction was collected by washing wells twice with cold 05 M glycine buffer (pH = 2.8) followed by a PBS rinse at 4°C. The internalized fraction was collected by lysing cells with 1 M NaOH followed by two PBS washes. All fractions were then counted on a Wizard2 automatic gamma counter

Purpose: Targeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation.
Procedures: To produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts. [1]

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Subcutaneous tumor model:[1]
Female athymic nude mice were injected with 5x106 BxPC-3 or MiaPaCa-2 cells in 150 μL (1:1 cell media:matrigel) in the hind flank. Tumors were allowed to grow for 3–4 weeks before imaging and biodistribution studies.

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Orthotopic tumor model:[1]
For orthotopic pancreatic xenografts, female athymic nude mice were used. Mice were anesthetized with 1–2% isoflurane and surgeries were conducted on a heated pad to regulate body temperature during the procedure. Bupivicaine was administered as a local anesthetic intradermally in the area near the incision. Skin around the incision was washed with 3 alternating wipes of povidone-iodine and 70% ethanol. An incision through the skin and peritoneum was made, followed by removal of the spleen and pancreas from the peritoneal cavity. At this time, 6 × 105 BxPC-3 (luciferase-transfected) cells in media and Matrigel (1:1 ratio) were injected into the head of the pancreas. The spleen and pancreas were inserted back into the peritoneal cavity, which was then sutured with 4-0 Vicryl sutures. To close the skin, three sterile wound clips were inserted. Buprenorphine and meloxicam were administered immediately following the surgery. Meloxicam was administered 24 and 48 h post-surgery and wound clips were removed 7 days later. Tumor growth was monitored by bioluminescent imaging (IVIS Spectrum) with tumors reaching optimal size in approximately three weeks. Clodronate liposomes were obtained from Formumax and each mouse received a 200 μL intraperitoneal injection.

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ICP-OES analysis:[1]
Analysis was carried out using an Optima 7000 DV spectrometer. The harvested organs containing [89Zr]Zr-5B1-AuNP and [89Zr]Zr-IgG-AuNP were first digested with an aqua regia solution of HNO3(65%): HCl (35%) at 75°C overnight and then dissolved in adequate volumes of 5% HNO3 solutions to be within the calibration curve range (from ppm to ppb). Hydrogen peroxide was added to speed the digestion of the organic materials. Calibration solutions were prepared from certified stock of a gold single element solution. The instrument was calibrated using a six-point calibration curve between 0.01 and 5 ppm and checked by three QC samples at the low, middle and high points on the curve. The operating conditions employed for ICP-OES determination were 1,300 W RF power, 15 L.min−1 plasma flow, 0.5 L.min−1 auxiliary flow, 0.8 L.min−1 nebulizer flow, and 1 mL.min−1 sample uptake rate. Signals at a wavelength of 267.595 nm were monitored. The low limit of quantification was determined to be 0.06 ppm. The comparison between the radioactive biodistribution data and ICP data is meant to be qualitative, in that it shows the same pattern of uptake. The discrepancy noted here we believe is due to the process by which the ICP measurements happen. Depending on the volume of acid required to dissolve organs, some concentrations may be below the limit of detection of the instrument (please note that the error bars are quite large for the ICP data due to this fact). This is a limitation of this method when using whole organs, as the volume of acid required for complete dissolution can be quite high; as well, a low injected mass of NPs, because it is difficult to dissolve, can make detection difficult for some samples, especially from the liver.

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Biodistribution studies:[1]
Biodistribution studies were conducted by sacrificing mice at discrete time points after injection of antibody-nanoparticle conjugates. Relevant organs and tumors were harvested, weighed, and counted on a gamma counter. Biodistribution values are presented as the percent of the injected dose per gram of tissue and were calculated by including appropriate standards.

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PET/CT Imaging:[1]
Mice were anesthetized with 1–2% isoflurane and images were acquired on an Inveon microPET/CT instrument.

[1]. ImmunoPET Imaging of Pancreatic Tumors with 89Zr-Labeled Gold Nanoparticle-Antibody Conjugates. Mol Imaging Biol. 2021 Feb;23(1):84-94.

In this work we have demonstrated the ability to deliver specifically targeted antibody-labeled gold nanoparticles to both subcutaneous and orthotopic pancreatic xenografts. When utilizing the humanized antibody 5B1, gold nanoparticles accumulated in subcutaneous pancreatic xenografts bearing the target antigen at 24.0 ± 11.6% ID/g compared to 4.0 ± 1.2% ID/g for the IgG-labeled control. This accounts for a 6–8x increase in tumors that expressed the target antigen. In an orthotopic model, 5B1-labeled gold nanoparticles accumulated 4–7 times more in tumors than did the IgG-labeled controls. Further, the ability of clodronate liposomes to enhance imaging of orthotopic pancreatic xenografts has also been demonstrated. This work carries implications for the development of methods of tracking nanoparticle biodistribution in real time in vivo. By incorporating a PET-active radionuclide onto our nanoparticles, we could visualize and analyze the effects of an active targeting moiety and macrophage depletion strategies. This could in theory then be used for other modifications made to any nanoparticle system that needs to be evaluated in vivo for clinical applications. The incorporation of a PET nuclide can enable the noninvasive tracking of drug effects or other factors in the biodistribution of nanoformulations. Further, gold nanoparticles themselves provide advantages such as (a) in vivo safety, (b) easy modification, and (c) the ability to target both passively through localization to the tumor microenvironment and actively by tumor cells. Lastly, this study supports a pharmacologic strategy that uses clodronate liposomes to enhance the biodistribution of a gold nanoparticle-antibody conjugate in order to enable imaging by PET. [1]

C33H52N8O8S2

752.94

752.334

1222468-90-7

24983484

White to off-white solid powder

1.5

7

11

26

51

1140

0

HBAYEVATSBINBX-UHFFFAOYSA-N

InChI=1S/C33H52N8O8S2/c1-26(42)39(47)22-8-2-5-19-34-29(43)15-17-31(45)40(48)23-9-3-6-20-35-30(44)16-18-32(46)41(49)24-10-4-7-21-36-33(51)38-28-13-11-27(12-14-28)37-25-50/h11-14,47-49H,2-10,15-24H2,1H3,(H,34,43)(H,35,44)(H2,36,38,51)

N-[5-[acetyl(hydroxy)amino]pentyl]-N'-hydroxy-N'-[5-[[4-[hydroxy-[5-[(4-isothiocyanatophenyl)carbamothioylamino]pentyl]amino]-4-oxobutanoyl]amino]pentyl]butanediamide

p-SCN-Bn-deferoxamine; Berdoxam; 1222468-90-7; p-NCS-Bz-DFO; Berdoxam [USAN]; TMK6ND3QJH; UNII-TMK6ND3QJH; p-Isothiocyanatobenzyl-desferrioxamine;

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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