Research on Synthesis of Stearylamide HYNIC-Hydrazone

In the last years, microwave-assisted has contributed significantly to the development of new and efficient synthetic processes. In this context, recently our group has developed a simple and efficient microwave assisted synthesis of HYNIC analogues. The general synthetic approach used was by a method analogous to the route used by Teixeira et al. [Teixeira, V.; Cabral, P.; Porcal, W. Microwave-assisted solid-phase synthesis of nicotinylhydrazones for use in radiochemistry of technetium-99m, Arkivoc, Accepted 2018].In this work, we describe the microwave-assisted synthesis of stearylamide HYNIC-hydrazone 3 (Scheme x).

As part of our ongoing investigations in the development of new 99mTc radiopharmaceuticals, we set out to design a new and convenient method for synthesis of HYNIC-hydrazones under microwave conditions, with the aim of increasing yields and shortening reaction times (Scheme x). In comparison to the conventional heating procedure, microwave irradiation considerably accelerated the formation of HYNIC analogues.

To the best of our knowledge,no other publication based on this chemical modality of of stearylamide HYNIC-hydrazone has been applied before for labeling liposomes with 99mTc.

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Liposomes were prepared by the lipid lm hydration technique followed by extrusion, which could be characterized, demonstrating that they had a nanometric size. Liposomes were principally made from mixture of the PC lipid, and stearylamine HYNIC-hydrazone which contributes to the formation of less permeable liposomes and of cholesterol which acts as a stabilizing lipid.

Liposomes composed of lipids such as PC and cholesterol are the most preferred drug delivery systems because they show optimal circulation half-lives and have been shown to accumulate at the tumor sites.

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For further increase of the circulation half-time PEG surface coating was included by adding PE-PEG(5000): 1,2-Dipalmitoil-sn-Glicero-3-Fosfoestanolamina-N-[Methoxy(Polyethylene glycol)-5000].

99mTc-HYNIC-Liposmes and its peguilated analogues were easily obtained in a yield over 90%, being stable over time, even at 24 hours, the purity was maintained 80%, it's were purified by a PD-10 column and eventually both liposomes were of high radiochemical purity as it was demonstrated by the chromatographic quality control. Studies reports that to formulate 99mTc-HYNIC complexes with higher solution stability, ethylenediaminodiacetic acid, EDDA, has been used. In these studies, the labeling is stable for 12 hours in the reaction mixture but there were at least three isomeric forms in solution. All though it has been shown that using tricine as colligation increases the stability of the complex, has a better stability in vivo and reduces the amount of isomers (Liu Shuang., 2005).

Stability assays were performed in order to evaluate how briefly the radionuclide can be released from the liposomes and in this respect to ensure the efficient delivery of radioactivity at the target while tested in vivo. Furthermore, release of the radionuclide from the liposomes may result in an incorrect interpretation of the biodistribution results and low quality scintigraphic images. So, in order to investigate the possible transchelation of liposomes an incubation with the competing amino acid cysteine (Clemens Decristoforo and Stephen J. Mather, 1999) showed that radiolabelled conventional liposomes derivatized with HYNIC present a greater stability than HYINC- PEG Liposmes, thus demonstrating that the stability given by pegylation is merely in vivo.

An ideal pattern of biodistribution of radiolabelled HYNIC liposomes with 99mTc is the one, in which no radioactivity is found either in stomach and thyroid gland or in kidneys and bladder, these suggests that the reduced radionuclide is tightly attached to the liposome and it is not presents as 99mTcO4. In effect, during biodistribution studies in healthy mice, uptake by the reticulo endothelial system(RES) rich organs of the liver and spleen for both kind of liposomes at 4 and 24 h. Specially spleen uptake was significantly higher for 99mTc-HYNIC-Liposomes indicating there are rapidly captured by macrophage cells. This was also observed by the relatively low blood values of 99mTc-HYNIC-Liposomes from the early time point of 4h. In contrast to radiolabeled HYNIC-Liposomes, for 99mTc-HYNIC-PEG-Liposomes had at 12.2 %Act/g at 4 h post-injection which is greater than 2.5 %Act/g of non-pegylated liposomes.

In addition to PEG lipid content, another aspect that must be taken into account is the particles size, where several studies have shown that liposomes that smaller than 100nm are less recognized by the RES organs (Papahadjopoulos et al., 1991; Allen et al., 1989, 1991; Boerman et al., 1995). The size of the liposomes in the present study was slightly larger than 100nm according to light diffraction and polarized light scattering measurements. After the in vitro stability was studied, biodistribution was performed in melanoma bearing mice.

Melanoma is the ninth most common cancer in Europe(Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline - Update 2016 JO - European Journal of Cancer,VL - 63 IS - SP - 201EP - 217PY - 2016 DA - 2016/08/01/T2 -AU -Garbe, ClausAU - Peris, Ketty AU - Hauschild, Axel AU - Saiag, Philippe AU - Middleton, Mark AU - Bastholt, Lars AU - Grob, Jean-Jacques AU - Malvehy, Josep AU - Newton-Bishop, Julia AU - Stratigos, Alexander J. AU - Pehamberger, Hubert AU - Eggermont, Alexander M. SN - 0959-8049 DO.

Over the past 30 years, the incidence of melanoma and non-melanoma skin cancer has been increasing steadily, with higher rates in women than in men. The World Health Organization warns of the increase in the world in the number of cases of melanoma, a type of skin cancer that are suffering more and more people and of which 200,000 new cases are detected every year.

Due these reason and availability of tumor model, it was conducted the biodistribution in C57 black mice bearing melanoma tumor induced with B16F1 cells. Biodistribution of 99mTc-HYNIC-liposomes in melanoma-bearing C57 black mice, shows the same biodistribution pattern than for normal mice, with a high percentage of liver and spleen uptake at 4 and 24 hours. At four hours blood/tumor ratio was 0.4, and tumor/muscle ratio was 5, while at 24 hours tumor/muscle ratio increased to 8 and the tumor/blood ratio increased to 113, evidencing low blood values at this time point.

Biodistribution in melanoma-bearing mice at 4 hours post-injection of 99mTc-HYNIC-PEG-liposomes shows that blood activity was (11.4 ± 1.40), superior to its conventional analogues (2.59 ± 0.86), hepatic uptake is equally high (27%), remaining close to this value at 24 hours (26) intestinal uptake increases from 2.8 ± 0.6% at 4 h to 13.3 ± 1.2% at 24 h.The tumor /blood and tumor /muscle ratios of the pegylated liposomes at 4 hours were: 0.2 and 6.4 respectively, whereas at 24 hours the tumor /blood ratio was increased to 1.4 and a slight decrease in the tumor /muscle ratio was observed reaching a value of a 4.2. It seems like that both liposomes exhibited preferential extravasation and accumulation at the tumor site owed to enhanced permeability and retention effect (EPR). This can be explained because liposomal formulations were of size between 100 and 200nm. It is important to mention that there is a very good correlation between biodistribution data and images, especially in the case of not overlapping organs such as liver, spleen and bladder.

Another important parameter to study is the liposomal dose. Results from the preliminary in vivo investigation in C57 black mice showed striking similarities for studied liposomes in the biodistribution prole, it was reported the %Act profile due that in this way it could appreciate urinary excretion. For both liposomal formulations at 1 hour post injection, were observed a greater urinary elimination in the groups of mice injected with a higher blocking dose . At the same time for both, the 99mTc-HYNIC-liposomes and their pegylated analogues, were observed a high liver and spleen uptake for the control group, which may be due to the capture of Liposomes by Kupffer cells at liver and endothelial reticulum system.

Liver uptake at 3 hours post-injection at the control group was lower in conventional liposomes than in pegylated ones, which is consistent with what is reported in the literature (Eduardo A. Castro, 1999), where incorporation of the polymer PEG would prevent opsonization and subsequent capture in the Kupffer cells. A high blood depuration of 99mTc-HYNIC-liposomes for any of the administered doses of cold liposomes can be observed. Dose-dependent biodistribution study of 99mTc-HYNIC-PEG-liposomes at 1 hour, shows that hepatic uptake of control group was 1.7 and 2.4 times higher than the 0.75 and 1.5 mg group, respectively. A similar observation is made gathering to spleen, where the groups of 0.75 and 1.5 mg presented a 2.3- and 4.6-times lower spleen uptake than the control group.

It was observed that the increase of urinary elimination was more obvious than the decrease of hepatic uptake, although this last one is an indicative of an effective blockade. Blockaded groups shows increased intestine activity, which may be consistent with a hapato-biliary elimination.

Taken together these data, could be confirmed that there is another route of elimination in addition to the hepatic biliary one, that justifies the decrease of the activity in blood, suggesting a metabolism and elimination of fragments through the urine. which cannot be due to liposome excretion via this pathway because liposomes are too large for renal filtration (Petersen et al., 2012). These eliminations through the urine it could not be attributable to the excretion of free 99mTc only because via biodistribution and scintigraphic imaging we did not observe any signi?cant uptake neither in the stomach nor in the thyroid gland for both studied liposomes.

The ultimate goal of this study is the design and application of liposomes as melanoma imaging systems. Currently, 18F-FDG is the most commonly used imaging agent for melanoma staging and the identification of metastases in the clinic. Melanoma tumors are in a high metabolic state and tend to import and trap 18F-FDG. However, 18F-FDG is not a melanoma-specific imaging agent and also is not effective in imaging melanomas that have primary closer to 18F-FDG normal uptaking organs (brain and blad) (Dimitrakopoulou-Strauss A, Strauss LG, Burger C. Quantitative PET studies in pretreated melanoma patients: a comparison of 6-[18F]fluoro-L-DOPA with 18FFDG and 15O-water using compartment and non-compartment analysis. J Nucl Med. 2001;42:248-256) Radiolabeled liposomes through stearylamide HYNIC-hydrazone could be used to noninvasively confirm the identity of a tumor or be used in cases in which 18F-FDG uptake is not optimal.

Image analyses shows that primary flank melanoma tumors were clearly visualized by both 99mTc-HYNIC-PEG-liposomes and 99mTc-HYNIC-liposomes. Scyntigraphic image of 99mTc-HYNIC-PEG-liposomes compared with their conventional analogues, shown a slower blood clearance, in agreement with activity levels at 4 and 24 hours. It should be noted that the 24-hour tumor / blood ratio (1.4) is not as noticeable as for 99mTc-HYNIC-liposomes (113), being consistent with the hypothesis that pegylation prevents the elimination of liposomes from blood, reaching higher time of residence.

However, when comparing tumor/muscle ratio in biodistribution at 4 hours of 99mTc-HYNIC-PEG-liposomes respect to the non-pegylation formulation, it can be seen that ratio was superior (6.4 vs. 5 respectively) being also consistent with what is observed in the scintigraphic image, where the comparison of the ROI with the contralateral side is 4.3 whereas for the conventional ones was 1.5 at the same time point.

At the first stage of this work shows scintigraphy imaging as a robust tool to provide non-invasively, accurate, quantitative information on liposomes spatio temporal biodistribution. The novelty of this work has to do with the in vivo comparison of two different liposome formulaton based on a new ligand (stearylamide HYNIC-hydrazone),which has not been previously used for labeling liposomes. The presented results con?rm this hypothesis and allow us to focus on the optimization of radiolabelling yield and design of liposomes for melanoma tumors in vivo, highlighting its potential role in other solid tumors and for sentinel node procedures.


In summary, the surface chelating labeling method via HYNIC-stearyamine, allow higher in vitro and in vivo stability and almost similar to previously published studies. Nevertheless, with this labeling strategy tumor regions were delineated using scintigraphic imaging. Imaging evaluation of in vivo targeting properties as well as in vivo drug delivery studies based on radiolabelled liposomes by the use of stearyl amide HYNIC-hydrazone provide an effective approach to noninvasively monitor the development of the melanoma tumors in vivo.

Updated: May 21, 2021
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Research on Synthesis of Stearylamide HYNIC-Hydrazone. (2019, Nov 27). Retrieved from

Research on Synthesis of Stearylamide HYNIC-Hydrazone essay
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