The Role of Sonication for the Preparation of Infection-Seeking Radiotracer Sample Versus the Standard Method

 

Dr Alireza Doroudi¹, Ehsan Rezaee², Dr Seyyed Mostafa Saadati³, Dr Ali Kiasat³,

Dr Faramarz Ahmadi³, Behrooz Etesami⁴, Dr Mostafa Erfani⁵

¹Associate Professor, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

²Candidate for Pharm D, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

³Assistant Professor, Nuclear Medicine Department Golestan general hospital, Ahvaz Jundishapur University of Medical Sciences. Ahvaz, Iran

⁴Nuclear Medicine Department Golestan general hospital, Ahvaz Jundishapur University of Medical Sciences. Ahvaz, Iran

 

ABSTRACT:

The main aim of this approach is to assess the evaluation of novel green chemistry to produce the new developed [^99mTc/Tricine/HYNIC] UBI _29-41radiotracer in comparison to the boiling water bath method. The 740MBq (20 mCi) of radiotracer samples were reconstituted by sonication or standard methods. The stability of radio complex samples in saline solution was investigated up 24 h post the reconstitution of cold kits. The protein bonding and partition coefficient of radiotracer samples were analyzed. The radioisotope imaging has been undertaken in order to determine the bio distribution of radiotracer samples in the infected rats with Staphylococcus aureus bacteria. The ITLC and Radio-HPLC profiles indicated that radio complex samples could be successfully reconstituted by sonication. The significant difference has not been observed in the stability, partition coefficient and protein bonding tests of radio conjugate sample which were prepared by either two methods. All infected lesions could be localized with imaging. The accumulation of radiotracer samples at the infected region versus contra lateral healthy side were identical. The outcome of this investigation indicated the reconstitution of freeze-dried UBI _29-41with ^99mTc with sonication versus the boiling water bath method has the advantages like the shorter the reaction time, excellent yield, milder condition in comparison to the conventional method and environmentally benign. Green chemistry can be recommended as an alternative and reliable method for the preparation of infection-seeking radiotracer in clinical practice.

 

 

INTRODUCTION:

Several radiopharmaceutical agents have been developed as an infection-seeking radiotracer in order to detect infection lesion. Ubiquicidin (UBI_29-41), is a cationic synthetic antimicrobial peptide fragment that can be preferentially attached to the anionic microbial cell membrane at the septic lesions^1,2.

 

This peptide fragment has 1.69 KDa molecular weight and six positive charge residues and the structure of molecule is depicted in Fig 1. UBI_29-41has affinity to react with technetium 99m (^99mTc) radioisotope which is widely used for labeling in the nuclear medicine in clinical practice. Therefore, UBI_29-41 peptide molecule could be readily labeled with reduced ^99mTcdirectly (^99mTc-UBI _29-41) or by using coligand cross linking such as 6-hydrazinopyridine 3-carboxylic acid (HYNIC) and tricine [^99mTc/Tricine/HYNIC] UBI _29-41indirectly^3,4. Recently the new developed radiotracer has been examined as an infection-seeking radiopharmaceutical in order to distinct infection foci preferentially in the nuclear medicine. According to the literature, the imaging with ^99mTc-UBI _29-41 radiotracer has been demonstrated the promising results if the pathogen caused infection was susceptible to the antimicrobial peptide fragment ^5-7. In spite of considerable capability of new developed radiotracer to tag septic lesions, the imaging with radio complex is suffered from the time-consuming period for the reconstitution of cold kit with ^99mTc radioisotope in the clinical practice. It is highly desirable to reduce the reaction time for radio labeling in the nuclear medicine wards. Green chemistry or sonochemistry is a branch of chemical research dealing with the chemical and applications of ultrasound irradiation. The sounds with frequencies above 20 kHz are usually used in sonochemistry and which lie beyond the upper limit of human hearing. Ultrasound irradiation waves can induce chemical effects on the reaction system, such as generation of free radicals which augment the rate of reaction ^8,9. In addition to the above mentioned factor, the new developed technique may have other mechanical effects on the reaction system, such as increasing the surface area among reactants, accelerating dissolution rate, reduced the time period of reaction and increased the yield of reaction^10-13. The ^99mTc-Sestamibi complex sample has been successfully prepared by ultrasound irradiation technique with high yield and short reaction time in radiopharmaceutical work ^14-16. This investigation was conducted to assess the preparation of [^99mTc/Tricine/HYNIC] UBI _29-41 radio complex by ultrasound irradiation technique and analysis of stability and pharmacokinetic parameters such as partition coefficient and protein bonding of samples in which the samples were prepared by sonochemistry versus boiling water bath as conventional method.

 

 

Fig 1: The proposed structure of^99mTc-UBI _29-41

MATERIAL AND METHODS:

All chemical materials have been procured from Merck and Sigma. The chemicals and solvents were the highest purity and analytical grade and used without further purification. The freeze-dried UBI_29-41 kits and ⁹⁹Mo/^99mTc generators have been provided by Radioisotope Division of Atomic Energy Organization of Iran (AEOI). Technetium 99m as sodium pertechnetate was obtained from an in-house ⁹⁹Mo/^99mTc generator using 0.9% saline. The rats with 135±15 g were obtained from research center and experimental animal house of Ahvaz Jundishapur University of Medical Sciences.

 

Animal Study:

This study was approved by the ethics committee of Ahvaz Jundishapur University of Medical Sciences. All the ethics issues were considered based on the Ahvaz Medical University of Medical Sciences (AMUP) on animal experiments. A total number of 10 adults, male NMRI were acclimated to the conditions for one week before the experiment. The animals were kept in individually cages in an air-conditioned room at 24±1ºC with a 12 hours' light-dark cycle and were fed with standard pellet diet and had free access to water. The rats were randomly assigned into two main groups equally. The radio complex samples were administrated intravenously due to contra lateral tail. The radio conjugate samples were prepared by the new developed method allocated to one group of animal. The radiotracer samples were prepared by the conventional method allocated to the other group of subject.

 

The reconstitution of UBI_29-41 complex samples:

Each new developed of UBI_29-41 vialcontained, in vacuum, lyophilized and pyrogen free mixture of HYNIC- UBI_29-41 40 µg, stannous chloride dihydrate 40µg and tricine 20 mg. The reconstitution of vials was undertaken as follow: 0.5 ml 0f 0.9 % saline was added in an evacuated and allowed the mixture to preincubate for 5 min. The freshly eluted of 740 MBq (20 mCi) of ^99mTcO₄ in 0.5 ml was added to the vials. The vials were put in the lead shield container and shaked for 5 min. The vials were heated by the water boiling bath device for 15 min at 95°C or sonicated in the thermo stated bath(Elma, P= 95 w made in Germany). The vials were incubated in the ultrasound irradiation instrument at the different times and temperatures in order to determine the optimum condition for the preparation of radio conjugate samples with appropriate yields. Instant thin layer chromatography (ITLC) and radio high performance liquid chromatography (Radio- HPLC) assays have been performed to identify and measure the yield of radio labelling and radio impurities. The strips of silica gel (Merck) in 2 cm width and 10 cm length were used in ITLC analysis as the stationary phase. The radio conjugate samples were prepared by either two techniques applied 1 cm from the bottom of ITLC strips. The strips were allowed to dry at the room temperature and put them in the air-tight containers. Two different solvent systems were used as the mobile phase. By using 0.1 M sodium citrate (_PH= 5), the desired radio conjugate and reduced technetium ^99mTcO₂ remained at the spotting point and non-peptide bond ^99mTc and free ^99mTcO₄ traveled to solvent front, when the mixture of methanol and 1 M ammonium acetate in the ratio of 1:1 used the^99mTcO₂ remained at the spotting point and free ^99mTcO_4, non-peptide bond coligand and radio conjugate moved to the solvent from. Then the strips were removed from the air-tight containers and allowed to dry at the room temperature. The strips were cut ⅓ lower and ⅔ upper pieces. The radioactivity of each piece was counted by the propose collimator an energy peak centered a 140 keV with NaI(Tl) detector for 2 min. Percent of [^99mTc/Tricine/HYNIC] UBI _29-41 radio complex sample was calculated from the following equation: the percent of desired radio conjugate equal 100- (the total of % non-peptide bond ^99mTc coligand, ^99mTcO₂ and ^99mTcO4)Each experiment was carried out triple times. The mean percent yields of radiotracer and radio impurity species are depicted in table 1.

 

Table 1: The 740MBq^99mTcO₄^- (20 mCi) freshly elution solutions of^99mTcO₄^- were added to the [Tricine/HYNIC] UBI _29-4 vials and the mixtures sonicated at 30,50,70 and 90 ºC in the thermo stated bath (Elma, p == 95 W, f == 60 Hz, made in Germany) for 1,2,3,4 and 5 min Each reaction was carried out triplets and mean of [^99mTc/Tricine/HYNIC] UBI _29-41,^99mTcO₂ and ^99mTcO₄ with ^99mTc coligand stated. Each reaction was carried out triplets and mean of [^99mTc/Tricine/HYNIC] UBI _29-41,^99mTcO₂ and ^99mTcO₄ with ^99mTc coligand stated.

Temperature ºC	Percent of species	Time (min)
		1	2	3	4	5
30	99mTcO4 with 99mTc Coligand	16.5± 1.1	19.7± 3.4	18.2± 1.63	20.1± 2.7	21.6± 1.5
	99mTcO2	54.31± 4.14	47.8± 2.18	47.64± 1.26	43.1± 2.1	40.5± 3.8
	99mTc-UBI 29-41	29.19± 2.19	32.5± 1.19	34.16± 1.85	36.8± 3.1	37.9± 2.6
50	99mTcO4 with 99mTc Coligand	15.72± 0.84	4.55± 1.35	5.3±1.87	6±1.2	5.05± 1.13
	99mTcO2	26.18± 1.7	22.35±1.5	19.1± 1.33	18.2±1.56	18.25±1.4
	99mTc-UBI 29-41	58.1± 4.6	73.1±2.8	76.5±3.7	75.8±2.8	76.7±2.65
70	99mTcO4 with 99mTc Coligand	2.06±0.24	2.25±0.8	2.3±1.1	4.35±0.65	8.05±2.7
	99mTcO2	10.34	7.65±0.85	5.1±1.9	6.35±1.74	5.45±1.16
	99mTc-UBI 29-41	87.6±2.1	90.1±2.2	92.6±1.66	89.3±1.35	86.5±3.3
90	99mTcO4 with 99mTc Coligand	7.2± 1.3	6.3±2.1	7.9±1.65	9.4± 2.05	12.6±2.28
	99mTcO2	15.7±1.65	18.5±3.2	17.5±2.95	19.9±0.77	22.1±2.8
	99mTc-UBI 29-41	77.1±1.55	75.2±2.2	74.6±1.9	70.7±2.35	65.3± 2.77

 

 

Fig2:  .Radio-HPLC profile of [^99mTc/Tricine/HYNIC] UBI _29-41 radiotracer

 

The ITLC analysis was a preliminary investigation in order to find out the suitable condition from the aspect of reaction time and temperature to prepare the radio conjugate with efficient yield. The Radio-HPLC analysis was performed with analytical reverse-phase on a JASCO 880-PU intelligent pump HPLC system (Tokyo, Japan) equipped with a multi wave length detector and a flow-through Ray test-Gabi g-detector CC 250/4.6 Nucleosil 120-5 C-18 column from Teknokroma was used for HPLC. For radionuclide analysis of [^99mTc/Tricine/HYNIC] UBI_29-41 complex sample by HPLC, a volume of 10μl of the test solution was injected into the C-18 reverse-phase column and trifluoroacetic acid 0.1%/water (solvent A) and acetonitrile (solvent B) were used as a mobile in following gradient: 0 min A 95% (B 5%), 5 min A 95% (B 5%), 25 min A 0% (B 100%) and 30 min A 0% (B 100%), flow= 1 ml/min Fig 2.

 

The chromatographs of radio conjugate samples were prepared 1 h post the reconstitution of cold kits due to a: sonication b: boiling water bath methods. The retention times of Free ^99mTcO₄ and[^99mTc/Tricine/HYNIC] UBI _29-41 radiotracer are approximately 4 and 13 min respectively.  

 

As it has been indicated in table 1, the yield of desired radio conjugate was suitable, when the reconstitution of UBI _29-41 with ^99mTc was carried out for 3 min at 70 °C under ultrasound irradiation. Therefore, the preparation of [^99mTc/Tricine/HYNIC] UBI _29-41 radio complex samples was performed on the above mentioned condition by new developed technique in all studies. The stability investigation of radio conjugate samples was assessed as the following procedure. Twenty freeze- dried of UBI_29-41 kits have been chosen and divided into the equal group. The one group of freeze-dried kit was labeled by ^99mTc radioisotope due to sonochemistry as the new developed technique. The other group was reconstituted by ^99mTc radionuclide due to boiling water bath method as standard or conventional method. The stability of radio complex samples was investigated after 0.5, 2, 4, 6 and 24 h post the reconstitution of freeze-dried kits by Radio-HPLC assessment. The data have been obtained from these experiments were stated in table 2.

 

Table 2: The yield of Free ^99mTcO₄ and [^99mTc/Tricine/HYNIC] UBI _29-41) radio conjugate samples in saline solution have been analyzed by Radio-HPLC.

 

Fig3: The stability of [^99mTc/Tricine/HYNIC] UBI _29-41 in human serum

 

The mean yields of free ^99mTcO₄ and [^99mTc/Tricine/HYNIC] UBI _29-41) infection-seeking radiotracer have been measured by Radio-HPLC analysis. The top of each row is related to the radio conjugate samples (n= 10) which were reconstituted by sonication. The bottom of each row is related to the radio conjugate samples (n= 10) which were reconstituted by boiling water bath method.

 

Protein Bonding:

Protein bonding of radio conjugate complex samples were prepared by the new developed or standard methods undertaken as following procedure. 1 ml of freshly human serum albumin (purchased from the Iranian Transfusion Organization Tehran) was added to 100 µl of the final solution of radio complex sample in micro tube. The mixtures were gently shaked for 10 min and incubated at incubator at 37°C for 24 h. Then each sample was treated by 1 ml of ethanol and centrifuged at 500×g for 10 min at the room temperature and followed by decanting the supernatant from debris. The activity of each portion was quantified by gamma counter. The protein bonding of radio conjugate or radio metal transferred to serum albumin was measured by the activity of precipitate to the total activity of sediment and supernatant multiple 100. Aliquots were analyzed with Radio-HPLC to assess serum stability for radio complex sample Fig3. Radio-HPLC chromatograms have been obtained from the supernatant solution of radio conjugate samples in human serum. The infection-seeking radiotracer samples were prepared a: sonication b: boiling water bath methods.

 

Partition Coefficient:

The value of log partition coefficient (log P) of each sample test was calculated by following: the determination of specific activity of octanol as an organic phase and water as an aqueous phase. The amount of 50 µl of sample test was added to the mixture of 2 ml of octanol and 2 ml water in the test tube. The above mentioned mixture was vortexed for 5 min and left 15 min at the room temperature. Following centrifugation at 500× g for 10 min, the organic and aqueous phases were separated and counted in an automatic well-type gamma counter. Three aliquots of 50 µl were sampled from each layer and counted and mean activities from the organic and aqueous phases were measured for each sample tube. The log P was calculated by dividing of the octanol phase by that of the aqueous phase.

 

Imaging:

Staphylococcus aureus has been chosen to induce septic lesion in the rats. The bacteria sampling procedure has beenexactly undertaken according to the protocol that has been previously reported to the literature ^17,18. The radioisotope imaging has been performed 48 h post bacteria inoculation to the animals. Under briefly anesthesia with ketamine, the rat was placed in the restrainer apparatus and the 37MBq (1 mCi) [^99mTc/Tricine/HYNIC] UBI _29-41 complex sample was injected by the contra lateral tail vein. The radio conjugate samples were reconstituted by new developed technique or water boiling bath method. The anesthetized live rat was placed in a prone position with limbs spread out and fixed on the board with surgical tape for scintigraphy imaging. For all studies a single-headed camera (E-Cam, Siemens, USA) was used.

 

The scintigraphy investigation was performed 1 h post injection in all studies. Acquisition parameters were as follows: matrix size 256×256, Zoom factor ×3, anterior and posterior views for 5 min and energy window 140keVand reconstitution Method: filter back projection. Anterior and posterior static images were acquired using a large field of view gamma camera peaked to 140 Kev with a 15%window and a low-energy all-purpose collimator for 500 kilocounts per image. The gamma camera was positioned to the affected part (induced infection by S aureus) and contra lateral healthy side. Two criteria have been chosen for interpretation of radioisotope scintigraphy images.

 

First criterion was the visual inspection of the radiotracer uptake at the infection site to the contra lateral healthy side. Second object, by using available commercial software region of interest (ROI) was created on the affected foot as target and then second ROI was generated on the contra lateral unaffected foot as non-target in interior views. The radiotracer uptake at the target versus non-target was quantified. Therefore, the specific uptake of radio complex sample was calculated by dividing the count per pixel in target to count per pixel in non-target region. The background subtraction was not used in radioisotope analysis.

 

Statistical analysis:

The calculations of means and standard deviations were undertaken on Microsoft Excel for each test assessment in this approach.

 

RESULTS:

When a new method is suggested, a knowledge of its properties is mandatory to determine. The fundamental requirement is the accuracy and reproducible of the results have been meticulously assessed. ^99mTc radioisotope is widely used for the preparation of diagnostic radiopharmaceutical in clinical practice. Its popularity is related to the suitable half- life (t_½ = 6.03 h), ideal photon gamma radiation (140keV) and absence of beta radiation. In addition to the above mentioned factor, it is available as generator for nuclear medicine departments. It has potential capability to label a large variety of ligands in which are suitable for imaging and assessment of various desired organs.

 

The low concentration of ^99mTc radioisotope is used for radio labeling in nuclear medicine. The activity of 740 MBq (20 mCi) is corresponded to approximately 10 ^-9 M. Chromatography analysis is commonly used as an accepted and reliable method for the assessment of radio labeling efficiency. ^99mTc is usually present as ^99mTcO₄ form in the eluted solution that is obtained from ⁹⁹Mo/^99mTc generator.^99mTc in the ^99mTcO₄form has the highest oxidation state and cannot react with the unshared electron of ligand. Most ^99mTc- Radiopharmaceutical agents except pertechnetate-99m itself, are reconstituted by the reduction of pertechnetate-99m in the presence of ligand agent.

 

A reconstitution of ^99mTc- Radiopharmaceutical is usually performed by the addition of ^99mTcO₄ to a cold kit in routine clinical work.^99mTcO₄,^99mTc coligand and^99mTcO₂ were radio impurities produced during the radio labelling of [^99mTc/Tricine/HYNIC] UBI _29-41 radio conjugate. All radio impurity species were identified by ITLC. The desired radio complex samples and free ^99mTcO₄ could be readily detected by radio-HPLC analysis.

 

 When the radio complex samples (n=3) were prepared by the conventional method, the yield of ([^99mTc/Tricine/HYNIC] UBI _29-41,^99mTcO_2, total free ^99mTcO₄ and ^99mTc coligand were measured by ITLC, 94.2± 1.2, 3.45± 1.8 and 2.35± 1.24 respectively. The percent of free ^99mTcO₄ and desired radio conjugate sample were 3.45± 0.55 and 96.55±0.7 respectively by radio-HPLC analysis. Ultrasound waves have two direct and indirect effects. Ultrasound waves have low energy (20 KHz to 500 KHz to alter the electronic, vibration and rotational molecular states in the direct effect. Ultrasound waves induce the cavitation phenomenon in order to generate enough energy to alter the vibrational and rotational molecular states in the direct effect. Pressure wave cycle exceed the attractive forces of the molecules and cavitation bubbles are formed. Bubbles grow over a few cycles and suffer sudden expansion and finally bubbles gently collapse and release the energy. Sonication was carried out at 30,50, 70and 90ºC for 1, 2, 3, 4 and 5 min. As it is stated in table 1, ultrasound waves could not produce the radio conjugate samples with sufficient amount of yields without heating. The reaction temperature and time period of sonication had paramount effect on the efficiency of radio labeling. When the reaction was examined at different temperature, the yield was increased with increasing temperature and reached to maximum 92.6±1.66%. But when the temperature was raised to 90ºC, the yields decreasedto77.1±1.55%.

 

The radio labeling was carried out at 70ºC temperature due to sonication on the basis of results in which found in this approach. The time period of reaction was another important factor. The yield was increased from 87.6±2.1to 92.6±1.66 % at 70ºC when reaction time was increased from 1 to 3 min the yield of reaction enhanced remarkably. But when the reaction time was raised from 3 to 5 min, the radio labelling efficiently was again reduced. Therefore, the preliminary experiment indicated this matter if the reaction for radio labelling was carried out at 70°C for 3 min under sonication, the radio conjugate samples could be prepared with high yield and suitable for the clinical use. As it is stated in Fig 2, the Radio-HPLC profile showed the retention times of ^99mTcO₄ and [^99mTc/Tricine/HYNIC] UBI _29-41 were approximately 4 and 13 min respectively, when the radiotracer conjugate was prepared by sonication. These values of retention time were identical in which radiotracer complex sample was prepared by the conventional method. This matter revealed that the reactions could be leaded to a single radio conjugate. This achievement demonstrated that the radio conjugate complex samples could be successfully reconstituted by the new developed technique. The stability of reconstituted [^99mTc/Tricine/HYNIC]UBI _29-41 complex samples was investigated up to 24 h after reconstitution of cold kits in which prepared by the new developed technique in comparison to the conventional method. As it is shown in table 2, The yields of radio labeling by ultrasound irradiation technique at different times were very similar to the yields of radio-complex samples which were prepared by the standard method. The decomposition of the radio conjugate samples has not been observed in this period, suggesting its high stability in normal saline at room temperature. The radiotracer samples were prepared by the both mentioned methods, they were completely found to be stable in final radiopharmaceutical samples and their radiochemical purity were measured above 90% up 6 h post the reconstitutions by Radio-HPLC analysis. The partition coefficient and protein bonding were 0.711± 0.14 and 74.40±1.87 % respectively for the radio conjugate samples in which were prepared by sonication. These values were 0.67 ± 0.11 and 68.39±2.28% respectively for radiotracer samples in which were reconstituted due to the standard method.

 

The significant statistical difference has not been observed in these values between the radio conjugate samples in which were prepared by either two above mentioned techniques in this investigation. The stability of radio conjugate samples was checked in human serum at 37°C for 24 h post the reconstitution. Incubation of [^99mTc/Tricine/HYNIC]UBI _29-41 complex samples in human serum showed good stability and the radiochemical purity of radiotracer remained above 90% under physiologic condition Fig 3. Radioisotope imaging has been performed for better comparison of biodistribution of radio conjugate samples which were prepared by two methods.

 

All induced septic lesions could be readily identified by imaging in the rats Fig4. Images with good quality were obtained in each case and the quality of images did not change over the time. The accumulation of radio conjugate was observed at the infected site in all images. The target/ non-target ratio was 2.65 ± 0.16 in the rats that radio conjugate samples were prepared by ultrasound irradiation technique. This factor was 2.71± 0.28 that radio complex samples were prepared by the boiling water bath method and administrated to the subjects. This finding indicated that the radiotracer uptake of [^99mTc/Tricine/HYNIC]UBI _29-41 complex samples were prepared by the two above mentioned methods were very similar at the infection foci induced by S aureus in the animals.

 

Fig4: Radioisotope imaging

 

The scintigraphy analysis was undertaken 1h post injection of 37MBq (1 mCi) [^99mTc/Tricine/HYNIC] UBI _29-41 infection-seeking sample due to the lateral tail vein. The interior view image could be readily localized the local septic lesion which created by Staphylococcus aureus. The radio conjugate samples were prepared by a: sonication b: boiling water bath methods.  

 

DISCUSSION:

Radioisotope imaging differs from most other imaging techniques in that diagnostic imaging intervention show the physiological function of the system being investigated as opposed to traditional anatomical imaging techniques such as X-ray, computerized tomography and magnetic resonance ^19,20. The most intensively used radioisotope is ^99mTc in nuclear medicine departments. Its popularity is related partly to its ideal radioisotope and chemical characteristics for the radio labeling of different ligands. In addition to the above mentioned factors, it is available as generator ⁹⁹Mo/^99mTc for radiopharmaceutical works in clinical practice.^99mTc radioisotope is present as ^99mTcO₄ in eluted solution that is obtained from ⁹⁹Mo/^99mTc generator. The saline ^99mTcO₄ elute from the generator is introduced by syringe via a septum into a vial containing the ligand and reagents necessary to prepare the desired imaging radiotracer. The reconstitution of some cold freeze-kits can be readily performed by incubation at the room temperature ²¹. Several ^99mTc-Radiopharmaceutical agents like [^99mTc/Tricine/HYNIC] UBI _29-41require heating as a step in their reconstitution. Inadequate heating, caused by insufficient incubation temperature or insufficient incubation time, may not provide the necessary energy to drive the reaction to completeness and therefore results in unacceptably high amounts of residual, unreacted free^99mTcO_4. This dilemma has been reported for a variety ^99mTc-Radiopharmaceutical             agents ^22-24.

 

Conversely, excessive heating may produce gas pressure inside sealed vials sufficiently high to cause rupture of the septum, ejection of the stopper or breakage of the glass walls. The boiling water bath is commonly used as a heating source in nuclear medicine. The radio labeling of radiopharmaceutical agents is usually time-consuming due to the boiling water bath method. It is highly desirable to establish an alternative and valid method that the preparation of radiopharmaceutical agents can be readily undertaken under milder condition versus the conventional method. Micro oven technique was examined for the reconstitution of Sestamibi in the nuclear medicine. This radiotracer could be prepared with the suitable yield in comparison to the conventional method. The required time for the reconstitution of Sestamibi by ^99mTc was reduced to 10 seconds when the microwave technique was used. during short period of time versus the conventional method by the new developed technique. The microwave oven technique is not widely applied for the radiopharmaceutical work in nuclear medicine department. Because this modality was seriously suffered from the following precautions and disadvantages. The geometry of the samples is very important when the samples are placed in the microwave oven instrument. The high potential risk of sparking for the presence of metal cap of sample. Microwave instrument with digital control panel is suitable for setting short heating time, since it must be accurately set at the required heating period. Ant technical error in setting the instrument heating time below or beyond the predetermined time may be leaded the reconstitution of radiopharmaceutical kit rendered inappropriate for clinical usage. Any residual gas left in the head space of the vial could cause an ejection of the rubber stopper due to the excess steam built up the vial. In addition to the aforementioned factors, the loss of variation of microwave oven output and frequency related to extended use of the device must be inspected on a long-term usage in order to obtain the radiopharmaceuticals with high efficiency and radiochemical purity^25,26. Sonochemistry is widely used in a variety of branches chemistry. The reactions are carried out to lead for faster rate, milder temperatures and better yields when ultrasound irradiation technique is used. Ultrasound waves indirectly affect chemical reaction through cavitation phenomenon. Cavitation generated a vacuum, form bubbles which grow over a few cycles and collapse violently. The energy released by the collapse provides the driving force to carry out the reaction.^99mTc radioisotope in the ^99mTcO₄ form is present in the elution solution which is routinely obtained from in-house ⁹⁹ MO/^99mTc generators. Technetium possesses valence from 1^- to 7⁺, of which 7⁺ and 4⁺ for technetium to be the most stable ones. Most ^99mTc-Radiopharmaceutical agents except pertechnetate itself, are prepared by the reduction of pertechnetate in the presence of a ligand agent. It is mandatory the diagnostic kit has a reducing agent in its formulation. Stannous chloride is widely used as reducing agent in this regard for the manufacturing freeze-dried radiopharmaceutical kits. The reduction of pertechnetate can be effectively undertaken by stannous chloride.

 

The reduction of the highest oxidation state of^99mTc to the lower valence state can be performed. The reduced ^99mTc can beable to react with unshared electrons of ligands which are present in the ligand's structure. Therefore, the structure of ligand must have electron donor groups like oxygen, nitrogen, phosphorus or sulfur in order to form the bonding between the unshared electrons of ligand and the empty orbitals of ^99mTc.It is obvious the nature of this kind of bonding is not strong as covalent or ionic bonding. The reconstitution of [^99mTc/Tricine/HYNIC] UBI _29-41 infection-seeking radiotraceris facilitated at elevated temperature. The local created heating by sonication without heating could not be produced the radio conjugate sample with suitable yield. The outcome of this study indicated that two main factors are involved to produce the radio conjugate samples with good yields. These factors are the temperature reaction and the time period of sonication. The yield of radiolabeling was increased to92.6±1.66%when the sonication reactions were carried out at the 70°C for 3 min. If the temperature reaction was raised above 70°C and time reaction exceeded beyond 3 min, the yields of radio labeling were dropped off into the amount that radio conjugate complex samples were rendered inappropriate for imaging in clinical practice. The vial with lead shield and metal cap could be placed in the water bath of sonication device. The geometry of samples inside the ultrasound instrument and potential risk of sparking were not considered as the precaution factors when the new developed technique was applied for the reconstitution of infection-seeking radio conjugate sample. The reaction time was decreased considerably in comparison to the conventional method. The radio labeling reaction was carried at the milder condition versus the boiling water bath method. The potential ionization irradiation exposure to the staff are working in the nuclear medicine departments could be effectively decreased. Energy consumption can be saved by ultrasound irradiation technique versus boiling water bath method. The reaction may be considered as an environmental benign. The reconstitution of [^99mTc/Tricine/HYNIC]UBI _29-41 can be undertaken in any nuclear medicine departments and permitted a fast and reliable method to make infection-seeking radiotracer. The following precaution must be considered when the new developed technique intended to prepare the infection-seeking radio conjugate samples. Different sonication devices with varieties power are available in the markets. It is necessary to figure out the optimum conditions from the aspect of temperature and reaction time on the basis of power of instrument. It is obliged that the legal considerations of using the new developed technique must be judged and approved for clinical application of this method for the reconstitution of [^99mTc/Tricine/HYNIC]UBI _29-41 radiotracer.

 

CONCLUSION:

Our achievement indicated that green chemistry can be successfully opened the new pathway for the preparation of radiopharmaceutical kits in nuclear medicine. The [^99mTc/Tricine/HYNIC]UBI _29-41 could be reconstituted with high efficiency under ultrasound irradiation technique. Sonication is recommended as an alternative and reliable modality for the radiopharmaceutical agents that the preparations are time-consuming.

 

ACKNOWLEDGEMENT:

This study is part of Pharm-D thesis of Ehsan Rezaee. The authors have no relevant financial interests related to the material in this manuscript. They also have no conflict of interests to declare. This work has been carried out with financial support from Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The authors gratefully acknowledge boss and stuffs of Radioisotope Division of Atomic Energy Organization Iran for assistance, good cooperation and the other contributions.

 

Abbreviations:

Bq: Becquerel, Ci: Curie, Mo: Molybdenum, TcO₄^-: Pertechnetate, Tc: Technetium, UBI: Ubiquicidin.

 

REFERENCES:

1.     Beiki D et al. ^99mTc-Ubiquicidin [29-4], a promising radiopharmaceutical to differentiate orthopedic implant infections from sterile inflammation. Iran J Pharm

2.     Ostovar A et al .Pooled analysis of diagnostic value of ^99mTc-Ubiquicidin (UBI) scintigraphy in detection of an infectious process. Clin Nucl Med 2013;38:413-416

3.     Akhtar MS et al. Antimicrobial peptide 99mTc-Ubiquicidin 29-41 as human infection- imaging : clinical trial. J Nucl Med 2005; 46:567-573

4.     Gandomkar M et al. Clinical evaluation of antimicrobial peptide [^99mTc/Tricine/HYNIC] Ubiquicidin 29-41 as a human-specific infection imaging agent. Nucl Med Biol 2009; 36:199-205

5.     Nazari B et al. Role of ^99mTc-Ubiquicidin 29-41 scintigraphy to monitor antibiotic therapy in patients with orthopedic infection: a preliminary study Nucl Med Commun 2011;32:745-751

6.     Assadi M et al. Diagnostic value of 99m Tc-Ubiquicidin scintigraphy for osteomyelitis and comparisons with 99m Tc-methylene diphosphonate scintigraphy and magnetic resonance imaging. Nucl Med Commun 2011;32(8):716-723

7.     Shinto As et al. Clinical utility of 99Tc-Ubiquicidin (29-41 as an adjunct to bone scan in differentiating infected versus noninfected loosening of prosthesis before revision surgery. Nucl Med Commun 2017;38(4):285-290

8.     Riesz P, Berdahl D, Christman CL. Free radical generation by ultrasound in aqueous and nonaqueous solutions. Environ Health Perspect 1985;64:233-252

9.     Riesz P, Kondo T. Free radical formation induced by ultrasound and its biological implications. Free RadicBiol Med 1992;13(3): 247-270

10.   Khalaj A et al. N-alkylation and N-acylation of 2,4-dinitrophenylamine by ultrasound. Asia J Chem 2009;21(2):997-1001

11.   Allahyari S et al. Ultrasound assisted co-precipitation of nanostructured CuO-ZnO-Al₂O₃ over HZSM-5: effect of precursor and irradiation power on nanocatalyst properties and catalytic performance for direct syngas to DME. UltrasonSonochem 2014;21(2):663-673

12.   Safaei-Ghomi J, Akbarzadeh Z. Sonochemically synthesis of arylethynyl linked triarylaminescatalyzied by CuI nanoparticles: a rapid and green procedure for songashira coupling. UltrasonSonochem 2015; 22:365-370

13.   Mirza- Aghayan M et al. Ultrasound- assisted direct oxidation of benzyl alchohols catalyzed by graphite oxide. UltrasonSonochem 2016;32:37-43

14.   Doroudi A et al. Preparation of 99mTc-MIBI complex under ultrasound irradiation. J Radioanal Nucl Chem 2013;298:1185-1190

15.   Doroudi A et al. Clinical application of ultrasound for preparation of 99mTc-sestamibi complex. Ann Nucl Med 2015;29(3):295-301

16.   Doroudi A et al. The stability of 99mTc-MIBI (Sestamibi) complex samples which prepared under ultrasound irradiation technique versus boiling water bath method. J Appl Pharma Sci2016;6(11): 126-134

17.   Doroudi A et al. Efficacy of ^99mTc-Ciprofloxacin and ⁶⁷ Ga-citrate scintigraphy to discriminate infection foci induced by Staphylococcus aureus from sterile inflammation induced by carrageenan in rat. Iran J Nucl Med 2015;23(2):96-102

18.   Doroudi A et al. The evaluation of sensitivity and specificity of new developed ^99mTc-Ofloxacin radiotracer to discriminate infection induced by Staphylococcus aureus and sterile inflammation induced by carrageenan in rat's foot. Int. J PharmaSci Res 2017;8(4):1590-1597

19.   Kiasat A et al. The efficiency of new developed ^99mTc-ceftazidime radiotracer to distinguish infection induced by Staphylococcus aureus and sterile inflammation induced by carrageen an in the rat model. Int J Res Pharm Sci 2017;7(2):7-14

20.   Doroudi A et al. The evaluation effect of omeprazole effect on the sensitivity of ^99mTc-MDP bone-seeking to detect simulated closed fracture in the rat's foot. Iran J Nucl Med 2017;25(2):92-99

21.    Erfani M et al. Optimization condition in labeling of ofloxacin with ^99mTc and its biological evaluation in Staphylococcus aureus and Escherichia coli for infection imaging. Iran J Nucl Med 2013;21(1):1-6

22.    Haney TA et al. Physical and biological properties of a 99m Tc-sulfuocollid preparation containing disodium edetate J Nucl Med 1971;12(2):4-68 23

23.   Eshima D et al. Technetium-99m0sulfur colloid for lymphoscintigraphy: effects of preparation parameters. J Nucl Med 1996;37(9):1575-1578

24.   Gagnon A et al. Fast labeling of technetium- 99m-sestamibi with microwave oven heating. J Nucl Med Technol 1991; 19(2): 90-93

25.   Lima MJC et al. Preparation and evaluation of modified composition for lyophilized of [Cu(MIBI)₄]BF₄ for [99mTc] technetium labeling. Braz Arch BiolTechnol 2005;48:1-8

26.    Hung JC, Wilson ME, Gibbons RJ. Rapid preparation and quality control method for technetium-99m-2-methoxy isobutyl isonitrile (technetium-99m-sestamibi). J Nucl Med 1991; 32(11):162-168

 

 

 

Received on 17.09.2017         Modified on 24.10.2017

Accepted on 01.11.2017      © RJPT All right reserved