Comprehensive Analysis and Quality Assessment of Freeze Dried Methylene Diphosphonate Labelled with 99m-Technetium: An Extensive Comparison of  Biodistribution Study with Commercial Standards

doi:10.21203/rs.3.rs-3784875/v1

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Comprehensive Analysis and Quality Assessment of Freeze Dried Methylene Diphosphonate Labelled with 99m-Technetium: An Extensive Comparison of Biodistribution Study with Commercial Standards

https://doi.org/10.21203/rs.3.rs-3784875/v1

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Objective:Technetium-99m (^99mTc) is an important radionuclide used for the prognosis of bone scintigraphy labelled with a suitable kit.

Methodology: The present investigations focus on the aseptic preparation and application of the methylene di-phosphonic acid (MDP) kit on bio-organ after labeling with ^99mTc collected from a ⁹⁹Mo/^99mTc generator.

Result:Before going to bio-application of the labelled kit, radiochemical purity was determined by paper chromatography using the High Purity Germanium (HPGe) detector and the radiochemical purity of ^99mTc-MDP was found to be 99%. Furthermore, negative results were found for pyrogen and bacterial endotoxin tests; fungal growth was also absent. Radiation dose uptake in different organs of Wister rats was measured by a dose calibrator, and observed results indicate the highest accumulation of ^99mTc-MDP in the skull and skeletal part. This study also performed a comparative study model between laboratory and commercially prepared Monrol MDP kits. This study was performed as bio-application of laboratory prepared ^99mTc-MDP prior to commercial production of cold kit at radioisotope production division of Bangladesh Atomic Energy Commission.

Conclusion: Overall, the observations from this study are within the accepted range and provide prospect for commercial production of the kits for human administration.

MDP

99m Tc

Endotoxin

Iodometry

Hypercalcemia

Hydroxyapatite

For long days, nuclear medicine has been used in diagnostic, therapeutic and preventive sectors, but diagnostic sector is a prime application among those [1]. Nuclear medicine involves molecular imaging with the use of radiopharmaceuticals, and help the physicians to diagnose and treat different diseases specially tumor and cancers[2]. The definition of radiopharmaceuticals can be depicted as drugs which are radioactive and have a pharmaceutical effect that is used for diagnosis or therapy and causes no alteration of physiological response in the patient[3]. A radiopharmaceutical can be either radioactive elements like ¹³³Xe or labelled compounds like ^99mTc-labelled compounds (radiopharmaceuticals)[4]. Technetium-99m, the most commonly used radioisotope for labeling radiopharmaceutical, is usually extracted from a ⁹⁹Mo/^99mTc generator through the elution as ^99mTcO4-[2, 3]. [5] describes radiopharmaceuticals as either radioactive elements like ¹³³Xe or labelled compounds like ^99mTc-labelled compounds (radiopharmaceuticals). For numerous years, diagnostic purposes have been served by freeze-dried kits (cold radiopharmaceuticals) that are labelled with ^99mTc [4]. The production of these kits has been carried out under aseptic conditions[3]. To prevent microbiological contamination during all production stages, it is important to outline the manufacturing process. ^99mTc labelled complexes have been and are being used as bone imaging agents[6] and ^99mTc labelled with Methylene Diphosphonate (MDP) has the entrapping power of hydroxyapatite crystals in the mineral phase of the bone with the diphosphonate compounds[7].

To ensure the quality of formulated kits, various analytical techniques are employed, including paper chromatography, which determines radiochemical purity and efficiency of kits. Bacteria endotoxin and fungal growth tests must be conducted within a specific microbiological range to confirm the sterility of ^99mTc-MDP kits. Bio-distribution studies identify target organs and predict safety and efficacy, which includes pharmacology and safety studies. Subsequently, the present study conducted initial in-vivo experiments on rats and assessed the outcomes against a control and reference group. The aim of this investigation is the preparation of freeze-dried methylene diphosphonate (MDP), it’s quality control procedures, the biodistribution study of Technetium-99m (^99mTc) labelled with MDP (^99mTc-MDP) and subsequent comparison with a reference Monrol MDP kits.

Materials

All chemicals, reagents and solvents used in this study were of laboratory grade and received from Sigma Aldrich. The research was carried on MDP kits which were formulated by the freeze-drying method. ^99mTcO₄⁻ was obtained from ⁹⁹Mo-^99mTc generator produced at the Radioisotope Production Division of Bangladesh Atomic Energy Commission.

Preparation of MDP cold kits

MDP can be distinguished by the general formula H₂O₃P-X-PO₃H₂, where X= -CH₂ for MDP [8]. Independent of the substituents, phosphonates show a propensity to form oligomers, which can be avoided by adding antioxidants such as ascorbic acid. The cold kit formulation of MDP was prepared with sterilized equipment under aseptic conditions in a laminar air flow hood, pH was adjusted to 6.0; each vial contains 10 mg MDP, 0.2mg ascorbic acid and 1mg Stannous chloride hydrate following International Atomic Energy Agency’s ( IAEA) guideline described in technical report (IAEA series no-466). IAEA has established guidelines for the preparation of MDP cold kits to ensure their sterility, quality and safety.Freeze drying is a drying technique which has three steps, freezing, primary drying and secondary drying; moreover, this method is used for thermo-labile products[9]. Liquid formulation of MDP has some decomposition issue due to temperature and heat; that’s why its need to lyophilize by using Freeze drying technique[10].

Quality control

Quality control is paramount in the preparation of methylene diphosphonate’s lyophilized kits. Ensuring rigorous quality standards not only gurantees the reliability and accuracy of the kits but also safeguards user safety. Each of lyophilized preparations were subjected to quality control testing which included microbiological testing (e.g; bacterial and fungal growth test), percentage of Radiochemical purity (%RCP). [11]

Microbiological Testing

Bacterial endotoxin test

To detect bacterial endotoxin, known as pyrogen, TAL (Tachypleus Amebocyte Lysate) reagent of sensitivity of 0.5 EU/mL was used. For this purpose, TAL reagent was reconstituted following manufacturer’s instruction adding specific volume of pyrogen free water. Specific amount of reconstituted TAL reagent (0.2 ml) were dispensed into each of the test ampoules and equal amount of test samples were added to the ampoules. Positive control sample containing endotoxin, as well as, negative control without endotoxin were included to validate the test. The ampules containing the mixture were incubated at 37°C for 1 hour and the gel formation or colour change were observed and compared with positive and negative control for indicating the presence of endotoxin.

Fungal test

Fungal contamination was tested using Potato Dextrose Agar (PDA) which is specifically designed to support fungal growth. The PDA media were prepared using manufacturer’s instruction and sterilized by autoclaving at 121°C and 15 psi pressure for 15 min. Next, sterilized PDA plates were prepared by dispensing 20 ml PDA media in each petri dishes and allowed to solidify for 30 minutes. The process was conducted in the sterile condition (by UV radiation) under a laminar air flow hood. The solid agar media plates were inoculated with small portion of sample (0.1 ml) which was spreaded over the surface of the PDA plate using a sterile inoculation loop. Similarly, three other media plates were inoculated with 0.1ml fungal spore for positive control. Three media plates were kept as media control and three media plates as bench control. Then, the inoculated media plates were incubated at 25°C for 3 to 7 days for fungal growth.

Radio-labeling and Radiochemical Purity Determination

For radio-labeling, required amount of sodium pertechnetate (Na^99mTcO₄) (250 mCi/2–3 mL elution from ⁹⁹Mo/^99mTc generator) was added into a vial containing MDP kit. The solution was shaken and kept in rest for 20 minutes at room temperature for obtaining ^99mTc-MDP radiopharmaceutical. The radiochemical purity of ^99mTc-MDP was determined using paper chromatography systems [12, 13]. Whatman 3mm chromatography paper was used as a stationary phase and acetone and 0.9% sodium chloride solution were used as mobile phase to separate reduced ^99mTc (^99mTcO₂) and ^99mTc-MDP from pertechnetate on (^99mTcO₄ ⁻ ). A droplet was spotted on the bottom point of strips of whatman paper. The strips were promptly placed in a test tube, to which approximately 1 ml of acetone was added and developed until the solvent front had drifted to the top point of the paper. After that, the strips were dried and cut into equal segments: top and bottom. Each portion was then counted for gamma radiation. The same procedures have been performed with the saline solution.

Biodistribution studies

Biodistribution was carried out on Wistar rats, of averaging 250 gm in body weight. 0.2 ml of radio labelled ^99mTc-MDP was injected into the tail vein (caudal vein) of the rat with dose around 2.0mCi. Experiments were performed also with lower doses and animals were anaesthetized before sacrificing two hours after dosage administration and targeted organs were separated like the skull (brain), bones (femur, humerus, spines) as well as other soft tissues. Radiation dose uptake was measured by a Dose Calibrator (Veenstra Instrumentation 404). Animals taken for experiments were grouped into three. The first group was the ‘Control’ group; in this group of animals, only Tc-99m was injected. The second group was the 'Standard’ group; in this group MON. MDP KIT (10 mg lyophilized powder) from ‘Monrol’ was used for labeling with Tc-99m and then injected into animals. The third group belonged to this study ‘MDP’; in this group prepared MDP was used for labeling and then injected into animals.

The results of the tests for bacterial endotoxin and fungal presence have both yielded negative outcomes. In the bacterial endotoxin test, there was an absence of any detectable endotoxins, indicating a lack of contamination by bacterial byproducts known for their potential harm. Similarly, the fungal test produced a negative result, signifying the absence of fungal elements that could compromise the integrity of the tested sample.

The radiochemical purity [13] of the complex was determined by the separation of radioactivity into complexed (^99mTc-MDPcomplex), free (^99mTcO₄-), and reduced hydrolyzed technetium(^99mTcO₂) in paper chromatography.

From acetone, % of free^99mTcO₄⁻ =\(\frac{HPGe count for the top part}{HPGe count for top and bottom part} \text{x}100\)

From saline, % of reduced -hydrolyzed ^99mTcO₂=\(\frac{HPGe count for bottom part }{HPGe count for top and bottom part}x100\)

Percentage of Radiochemical purity = 100- (% of free^99mTcO₄ + percentage of ^99mTcO₂)

The radiochemical purity of the prepared kits is shown in Table 1

Table 1

Radiochemical Purity of ^99mTc-MDP
Sample No.	%^99mTc-MDP (RCP)	% ^99mTcO₄⁻	%TcO₂
1	99.93%	0.001	0.07
2	99.98%	0.0004	0.013
3	99.95%	0.003	0.038

Biodistribution is primordial to mark target organs and evaluate safety and efficacy, pharmacology studies, and anticipating based on specificities of vectors and constructs[14]. Radiopharmaceuticals are usually consist of two components, a radioactive element (radionuclide), that permits external scan, linked to a non-radioactive element and other component involves a biologically active molecule that depicts a drug or cell (red and white blood cells labelled with a radionuclide, for example) that acts as a carrier or ligand, responsible for conducting the radionuclide to a specific organ[15]. Because of this binding action radiation dose uptake was assembled in tissues, brain and skeleton of Wistar rats, that was measured by a dose Calibrator and Fig. 1 represents the percentage of radiation dose uptake in different organs concerning whole-body uptake. Less accumulation was observed in the control group than in the MDP group. The highest uptake was measured in MDP 2, standard, MDP 1 and control groups respectively for the skull (18.23, 16.04, 16.17 and 7.23%) and femur (15.28, 13.52, 7.84, and 2.41%) but not in skeletal muscle (In Fig. 1, percentage of flesh). Other groups can be summarized as having maximum similarity in the accumulation of ^99mTc-MDP( Fig. 1)

Accumulation in the femur in the standard group is > 13% whereas for MDP 1 is approximately 8% and for MDP 2 is up to 15%; the control group has accumulation in the femur is > 2% (In Fig. 2). The obtained results indicated the highest accumulation of ^99mTc-MDP in the brain and skeleton that may be associated with binding to hydroxyapatite crystals and calcium salts moreover. Uptake in soft tissue e.g. lungs, kidney, liver, pancreas intestine (especially in the control group) occurred may be due to a sort of pathological condition like hypercalcemia[16]. The assembling mechanisms of Technetium-99m with methylene diphosphonate (^99mTc-MDP) were scrutinized using hydroxyapatite powder and various phosphate in bone tissue[17]

Skeletal part ingestion is partly delineated in Figure (2), in which it is observed that the maximum dose was accumulated in the skull (brain part) and femur > 16%. Several studies summarized that about 50% of the radiation is retained in the skeletal part and the remaining is distributed in other body part[18]. This research also attained a similar sort of result,that illustrates maximum accumulation in skeletal parts. Ingestion of ^99mTc-MDP in higher amounts in the skull may be explained as the presence of small bones in the nervous system structure that produce enhanced surface area[19] as well as maximum absorption and this finding on bone scintigraphy can be influential in the diagnostic arena or the clinical assessment of the patient[20]. Furthermore, ^99mTc labelled MDP and other compounds are used as imaging agents of the central nervous system nowadays [21]. There is another technique radiographic skeletal survey which is more sensitive than bone scintigraphy because it tends to exhibit old fractures that have healed. On the other hand, it is quite difficult in bone scintigraphy and skeletal survey is kept reserved for rare cases[22].

The correlation between organs and the percentage of injected dose per organ cannot be defined perfectly yet, but there are many investigations about radio sensitivity to the weight of different organs. The radio sensitivity of the organs is influenced by the type of organs, the physiologic condition of these organs, size and weight of the organs. The group of stem cells that may or may not be replicating or proliferating, demonstrates the radiation persuade sensitivity of the organ enhanced by assembling many weights by having cells of different features. It is detectable that an increase in organ weight has clinical outcomes [20]

According to that statement, it was observed that the amount of persuaded dose is related to the weight of organs and that is delineated in the above picture (Fig. 3); the interrelationship between the weight of various organs and the percentage of corresponding induced dosage. The percentage radiation dose uptake in skeletal parts per organ weight in the present study groups (standard > 0.8, control > 0.5, MDP1 > 8.5 and MDP2 > 0.76 in the skull area) which is manifested in the above picture (Fig. 4) such as the femur and other bones (humerus, skull, spines) had shown maximum ingestion of radiation dose for MDP 1 group, respective to other groups. Maximum absorption of radiation dose in bones ( femur, humerus, skull) may be explained as bone contains a larger mass of calcium than soft tissue[16]and extended content of bone nutrients as well as the binding to regions of active bone metabolism [23]. The background chemistry of these systems is very complicated and there is no consistent theory that can explain the in-vivo mechanism of action but it can be informed that the interaction between diphosphonate ligand and Technetium-99 is possible mode of this action[24]. Another study was carried out that referred to the contribution of Sn²⁺ ions in the accumulation of 99m-Technetium- MDP, where Sn²⁺ ions act as reducing agents that permeated conversion of pertechnetate (^99mTcO₄⁻) to technetium (III) (^99mTcO₃⁻), furthermore, formation of lipophillic ^99mTc-Sn(OH)²⁺ complexes, that crosses blood brain barrier due to lipid solubility and allowing them into ‘calvaria’ which means the top part of the brain.[25]

It appears from the above discussion that percentage of induced dose or uptake of radiation may be dependent on bone infrastructure, number of bones and organ weight. It is also vital to consider the physiologic and pathologic condition of the patient, disease state, metabolism rate, diffusion of oxygen from the blood to tissue and functions of various tissues. However, it would be better to conclude the result from human autopsy sampling because the animal model is a better approach to finding a better insight of this present research.

The novel formulation, methylene diphosphonate acid and complex ^99mTc-MDP have been successfully prepared. The radiolabeling efficiency of the complex is high and its stability duration is long enough to allow biodistribution and imaging studies. Compared with the complexes ^99mTc-MDP (MON. MDP KIT 10 mg) and control (^99mTc) displays highly selective uptake in the skeletal system and uptake in the soft tissues (occasionally). These satisfying results lead us to further investigate it as an imaging agent for bone scintigraphy by commercial production.

Conflict of Interest Statement

All the authors have no conflict of interest

Funding Statement

This Research was supported by Bangladesh Atomic Energy Commission

Acknowledgement

The authors would like to express their sincere gratitude to the Radio Isotope Production Division, Institute of Nuclear Science and Technology in Atomic Energy Research Establishment, and all those who contributed to the preparation and submission of their research article. Special thanks go to their co-authors for their valuable insights and collaborative efforts. The authors also acknowledge the support of their institutions and all researchers who have been instrumental in this research endeavour and also thankful to the Bangladesh Atomic Energy Commission for its financial support throughout the study.

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Comprehensive Analysis and Quality Assessment of Freeze Dried Methylene Diphosphonate Labelled with 99m-Technetium: An Extensive Comparison of Biodistribution Study with Commercial Standards

Status:

Version 1

Abstract

Figures

Introduction

Experimental

Preparation of MDP cold kits

Quality control

Microbiological Testing

Bacterial endotoxin test

Fungal test

Radio-labeling and Radiochemical Purity Determination

Biodistribution studies

Result and Discussion

Conclusion

Declarations

Conflict of Interest Statement

Funding Statement

Acknowledgement

References

Status:

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