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Preparation, Quality Control and Biological Evaluation of 99mTc-Clarithromycin in Animal Models for Detection of Bacterial Infection.

Byline: Saira Hina, Samina Roohi, Muhammad Ibrahim Rajoka, Tanveer Hussain Bokhari, Muhammad Sohaib and Asma Haque

Summary: Clarithromycin is a macrolide antibiotic effective against bacteria by interfering their protein synthesis. In the present investigation, 99mTc-clarithromycin as a promising radiopharmaceutical for detecting staphylococcus aureus infection imaging in nuclear medicine is evaluated. At pH 7, clarithromycin was labelled with 99mTc with a labelling yield of 99% by adding 99mTc to 500 ug of clarithromycin in the presence of 25 ug using stannous chloride as reducing agent at room temperature. Radiochemical purity of the compound was analyzed by ascending chromatographic techniques and HPLC analysis. Electrophoresis indicates the neutral behavior of 99mTc-clarithromycin.

HPLC analysis confirms the single species of the 99mTc-clarithromycin complex. Biodistribution of 99mTc-clarithromycin was evaluated in animal models by inducing infection using live S. aureus, heat killed S. aureus, and turpentine oil (sterile inflammation model) which showed high uptake in infection induced sites (live S.aureus). Bio-evaluation by scintigraphic imaging also revealed high uptake of 99mTc-clarithromycin at S. aureus infected sites. Biodistribution and scintigraphic imaging of 99mTc-clarithromycin in infected mice and rabbit showed a good target- to-non target ratio indicating it a possible bacterial infection imaging agent.

Key Words: Clarithromycin, Bacterial infection, Technetium-99m, Staphylococcus aureus, Biological distribution, Scintigraphic Imaging.

Introduction

Bacterial infection and inflammation is a universal problem particularly in developing countries resulting in sickness, disability or mortality [1]. Inflammation is the reaction of tissue to any injury carrying cells of the immune system and serum molecules and to the area of tissue injury while Infection is developed by any injury caused by microorganisms [2]. Infection/ Inflammation diagnosis at very early stages is a major task for medical practitioners these days. The identification and localization of infection locus is very crucial for early diagnosis and medical treatment of the patients under clinical trials. Assessment and exact detection of infectious foci by means of imaging methods such as computerized tomography, MRI and ultrasonography is applicable but because of insignificant morphologic variations at early stages of diseases these practices do not contribute significantly [3, 4].

Nuclear medicine is very important technique for detection of infectious and inflammatory conditions by the use of whole body imaging permitting the localization and degree of disease activity in infectious processes. The radiopharmaceuticals seem like to resolve the issue of specificity by several mechanisms directly at site of infection or adhere to cells that successively migrate to septic foci [5-6]. One tracer which shows potential is Technetium-99m (99mTc) labeled anti-infective agent which has recently been introduced as infection seeking tracer [7]. Technetium-99m (Half-life: T 1/2 = 6.7 h) widely eluted from Mo-99/Tc-99m generator systems in all the radiopharmacy centers where medical interesting radiopharmaceuticals are devloped for gamma imaging.

Several 99mTc-labeled compounds such as, 99mTc-ceflizoxime [8], 99mTc- lomefloxacin and99mTc-ofloxacin complexes [9], 99mTc- enrofloxacine [10], 99mTc-erythromycin[11], 99mTc- cefuroxime[3], 99mTc-Kanamycin[12], 99mTc- ciprofloxacin[13,14], 99mTc-vibramycin[15], 99mTc- pefloxacin[4], 99mTc-ethumbutol[16], 99mTc- infecton[17], 99mTc-flucanazole [18], 99mTc- vancomycin [19], 99mTc-piroxicam [20], 99mTc- tetrofosmin [21], 99mTc-azithromycin [22], 99mTc- moxifloxacin [23], 99mTc-Rufloxacin [24], 99mTc- clindamycin [25] have been developed for infection imaging purposes. Clarithromycin is the structural analogue of erythromycin used extensively for the treatment of a variety of bacterial infections. Clarithromycin is an advance macrolide antibiotic characterized by a macrocyclic lactone ring to which deoxy sugars are attached. It is effective against gram positive bacteria by interference in their protein synthesis [26-30].

The main aim of current study was effective labeling of clarithromycin with 99mTc (a medical tracer) to produce a compound that is able to disrupt the protein synthesis of bacteria and at the same time identify its location. The evaluation of 99mTc-clarithromycin was done by chromatographic analysis and bio-evaluation was performed by means of biodistribution and scintigraphy thus making it available as a potential bacterial infection imaging agent.

Experimental

Drugs and Chemicals

Clarithromycin injection was the product of Abott Healthcare Products Limited. All reagents used were of AR grade and obtained from E. Merck, Germany. Na99mTcO4 was eluted with 0.9% saline from PAKGEN 99Mo/99mTc generator. S. aureus (ATCC 25923) was obtained from NIH, Islamabad. Radioactivity of tissue and organs was measured with a g-counter (Ludlum model 261). The scintigraphic imaging of rabbits was performed by using Gamma scintillation camera.

Animals and Ethics

For biodistribution study Swiss Albino mice weighing 30-35 g were obtained from National Institute of Health (NIH), Islamabad and were given free access to food and water. Animal experiments were followed by the standard rules and procedures of laboratory animal care and approved from the Animal Ethics Committee of the Pakistan Institute of Nuclear Science and Technology (Document no. IPDs-H-SOP-04-003).

Synthesis of 99mTc-clarithromycin

The experiments were performed to determine the optimum conditions for the synthesis of 99mTc-clarithromycin. The different amounts of clarithromycin and reducing agent (SnCl2.2H2O) were used. The pH of the reaction mixture was adjusted by the use of 0.5 M NaOH. After adding all reagents the mixture was stirred for few minutes and freshly eluted ~ 330 MBq Na99mTcO4 in physiological saline (0.9% NaCl) was injected into the vial. The volume of reaction mixture was ~1.5 mL. All experiments were performed under sterile conditions at room temperature.

Radiochemical Analysis of 99mTc-Clarithromycin

To determine the Radiochemical purity of 99mTc-clarithromycin, the sample of 1 uL was spotted at ITLC silica gel strips (Gelman Laboratories) and developed using 0.5 M NaOH as the mobile phase. By using this method, 99mTc-clarithromycin and free pertechnetate migrated with the solvent front of the mobile phase of the strip and the colloid was found at the origin. For determining 99mTcO -, a strip of Whattman Paper No. 3 was developed by using acetone as mobile phase. By the use of this system, 99mTcO4 migrated with solvent front of the mobile phase while labeled/reduced hydrolysed 99mTc remained at origin. The scattering of labeled, free and hydrolyzed on chromatographic strips was determined by the use of 2p Scanner (Berthold, Germany).

Electrophoresis study of 99mTc-clarithromycin

A drop of 99mTc-clarithromycin was spotted on Whatman No. 1 paper strip of 30 cm and PBS (pH 6.8) in an electrophoresis bath (Deluxe electrophoresis chamber system by Gelman). The voltage of 300 V was applied for 30-60 min at the room temperature. The strip was dried after ending electrophoresis and scanned by 2p scanner for the charge on99mTc-clarithromycin.

HPLC analysis of 99mTc-clarithromycin

High performance liquid chromatography (HPLC), equipped with NaI crystal detector and UV detector (wavelength of 254 nm), was done on an analytical reverse phase column of C-18 (Alltech) as stationary phase. The mixture of acetonitrile and 0.02M Sodium dihydrogen phosphate (pH adjusted up to 2-3 by using 0.5M NaOH) were used as mobile phase in the ratio 850:150 (v/v %) at a flow rate of 1mL/ min.

Human Protein Binding Assay

The human serum stability of 99mTc- clarithromycin was analyzed in vitro by addition of 1.8 mL of normal human serum with 0.2 mL of 99mTc-clarithromycin and incubated at 37oC for 24 hours. 0.2 mL aliquots were taken out at 1h, 4h and 24h and analyzed by instant thin layer chromatography for determination of 99mTc- clarithromycin, hydrolyzed/reduced Tc-99m and free 99mTcO4-.

Bacterial Strains

S. aureus (ATCC 25923) was acquired from the American Type Culture Collection (Manassas, VA, USA). The cultures of S. aureus were grown overnight in brain heart infusion broth (BHI, Oxoid) in a shaking water bath at 37oC. Suspensions comprising viable stationary phase bacteria were frozen in liquid nitrogen and kept at -70oC. Just before starting the experiment the aliquot of suspension was liquefied in a water bath at 37oC and diluted in PBS.

In vitro Bacterial Binding studies

In vitro binding of 99mTc-clarithromycin can be used for accessing infection induced by S. aureus using procedure reported [31]. Tc-99m labeled clarithromycin (~6 MBq) in 0.1 mL of Na-PB was transmitted in a sterilized test tube. After this 0.8 mL of 50% (v/v) of 0.01M acetic acid in Na-PB containing almost 1x 108 viable bacteria were added and incubated at 4oC for 1 hour followed by centrifugation for 10 minutes. The supernatant was separated and pallet of bacteria was suspended in 1 mL of ice cooled Na-PB. The supernatant was again separated and radioactivity in the bacterial pallet was determined by using g-counter. The activity in supernatant was counted also. The radioactivity bound to bacteria was stated in % of the added Tc-99m activity attached to viable bacteria compared to total Tc-99m activity.

Experimental Infection Models

Staphylococcus aureus from biological samples was employed to induce focal infection. Individual colonies were diluted to attain the turbid suspension of S. aureus. About 2x108 colony forming units of S. aureus in 0.2 mL of saline were introduced intramuscularly into the left thigh muscles of mice [32 33]. Mice were than kept under observation for 24 h to get a visible swelling in infected thigh. Sterile inflammation was brought by the injection of 0.2 mL of turpentine oil intramuscularly in the left thigh muscle of the mice [34]. After two days the swelling appeared. Sterile inflammation with the help of heat killed bacteria was induced by injecting 0.2 mL of heat killed S. aureus intramuscularly in the left thigh muscle of the mice. The swelling became visible after 2 days.

The S. aureus suspension was provided the temperature of 100degC for 2 h for heat killing and turpentine oil was sterilized by means of autoclaving at temperature of 121degC for about 20 minutes. The sets of three mice were used for all experiments.

Biodistribution of 99mTc-clarithromycin in mice

The Swiss Albino mice were injected intravenously with 0.2 mL of 99mTc-clarithromycin (~38 MBq) intravenously via tail vein. Then, the mice were sacrificed at 30 min, 1h, 4h and 24h post- injection after ether anesthesia. Samples of the different organs were separated and counted for radioactivity distribution in a well type gamma counter. The average percentage values of administrated dose per tissue were calculated. By cardiac puncture 1mL blood was taken, weighed and activity was calculated by supposing blood volume 6.34% of the body weight. The study was approved and was in accordance with the guidelines determined by animal ethics committee.

Scintigraphic Imaging of Tc-clarithromycin in Infected Rabbits

The evaluation of 99mTc-clarithromycin as potential infection imaging agent was further confirmed by scintigraphic study in rabbit models by single headed Siemens Integrated Orbiter Gamma Camera System interfaced with high-resolution parallel hole collimator. The infection was induced intramuscularly by injecting 0.4mL of S. aureus containing 2x108 colony-forming units in saline in left thigh muscle of rabbit. After ethical approval, animal was placed on a flat hard surface fixing the legs with the help of a surgical tape. After injecting Diazepam (5 mg) into the right thigh muscle, intravenous injection of 74 MBq of 99mTc- clarithromycin was given into the marginal ear vein. Immediately after injection, imaging was done with both thighs in focus.

Results and Discussion

Synthesis and Radiochemical Quality control

The use of targeting pharmaceuticals as a promising approach for early detection of infections has been developed in recent years. Clarithromycin is a potent macrolide antibiotic employed for the treatment of different bacterial diseases. In this experiment we have established a simple and efficient method of labeling clarithromycin with Technetium-99m (a medical tracer) that emits safe levels of gamma rays which can be detected with gamma camera. This technology uses the special antibacterial property of clarithromycin and the marking property of 99mTc to produce a complex for treatment as well as imaging purpose. The structure of clarithromycin is shown in Fig. 1.

The labeling efficiency of 99mTc- clarithromycin was evaluated by instant thin-layer chromatography and ascending paper chromatography on silica gel. In current study, clarithromycin was labeled with 99mTc with high radiochemical yields. The influence of amount of reducing agent (SnCl2.2H2O) on the labeling efficiency of clarithromycin with Tc-99m is shown in Fig. 2. The maximum labeling yield of 99mTc- clarithromycin was observed at 25 ug and as the amount of reducing agent was increased the labeling efficiency reduced. In order to determine the most appropriate pH for the labeling, the labeling of 99mTc- clarithromycin was carried out at different pH in range from 3-11. At pH 7complexation rate of clarithromycin with Tc-99m was maximum (>99%). labeling yield of 99mTc-clarithromycin was decreased to ~42% in acidic conditions (pH 3). In basic conditions, at pH 11 the radiochemical yield reduced to 78% (Fig. 3).

The influence of amount of ligand having the highest labeling efficiency was checked by labeling of varying amounts of clarithromycin with 99mTc and the amount of 500 ug ligand was observed to show high yield of labeling as shown in (Fig. 4). After 30 min the maximum labeling of clarithromycin with 99mTc was achieved at room temperature (Fig. 5).

The radiolabeling of clarithromycin with 99mTc resulted 99+-0.4% radiolabeling yield. The results of optimization of affecting factors on radiolabeling showed that at reaction condition of 500 ug clarithromycin, 25 uL SnCl2.2H2O, pH=7, and 30 minutes incubation time, the labeling yield of clarithromycin reached 99+-0.4%.

Paper Electrophoresis and HPLC analysis

The electrophoresis results demonstrate the neutral nature of ligand and the free pertechnetate movement was observed towards the anode after 1 hour (Fig. 6). The HPLC anlysis of 99mTc- clarithromycin is given in Fig. 7 which shows a very sharp peak at 1.7 minutes of retention. This signal at 1.7 minutes represents the 99mTc-clarithromycin labeling efficiency >99%. The HPLC analysis by UV detector (inactive ligand) show percent purity of clarithromycin (Fig. 8).

In Vitro Binding with S. aureus

The binding of 99mTc-clarithromycin to S. aureus was in the range of 70-76% as represented in Table-1. The amounts of 99mTc-clarithromycin in the range of 10-50 ug indicated similar binding effectiveness with bacteria while as the amount was increased to 100ug binding efficiency was decreased. In vitro Incubation of 99mTc-clarithromycin with the bacterial strain S. aureus has demonstrated a high bacterial retention of tracer. About >70% of the total activity was accumulated in bacterial pellet of S. aureus after incubation with 99mTc-clarithromycin in a bacterial culture. The values found in present study are >those obtained by other studies. The uptake of Tc- clarithromycin was found to be >99mTc-azithromycin (47-65%) [24].

Table-1: In vitro binding of the 99mTc-clarithromycin to live Staphylococcus aureus

Incubation###99mTc###Free

###Colloid

time, h###clarithromycin###pertechnetate

###1###98.9 +- 1.2###1.1 +- 0.5###0.0 +- 0.4

###2###98.7 +- 1.1###1.3 +- 0.8###0.0 +- 0.6

###4###97.1 +- 2.0###0.9 +- 1.1###2.0 +- 0.7

24###97.0 +- 1.9###2.2 +- 1.4###0.8 +- 0.9

Stability in Human Serum and Room Temperature

99mTc-clarithromycin was completely stable in human serum during incubation as determined by ITLC. It was found that 97% labeling was found at 24 hours of incubation with very little increase in free 99mTcO4 and reduced or hydrolyzed 99mTc at 37oC. The total impurities found were that of other reported 99mTc-labeled antibiotics, for example 99mTc-azithromycin (T/NT = 6.20 +- 0.12) [22], 99mTc-erythromycin (T/NT = 5 +- 0.6) [11], difloxacin (5.5 +- 0.5) [39], sparafloxacin (5.9 +- 0.70) [40], ceftraixone (5.6 +- 0.6) [41], N-sulfanilamide (2.9 +- 0.1) [42], and streptomycin (2.4 +- 0.1) [43].

Mice having infected sites injected with 99mTc- clarithromycin showed a good T/NT ratio, which are in agreement to the results obtained from the in vitro binding study. The greater activity of 99mTc- clarithromycin in bacterial infection sites than inflammation sites can be attributed to specific binding to living bacteria in mice.

Table-3: Biodistribution of 99mTc-clarithromycin (%ID/g) in live S.aureus, heat killed S.aureus and turpentine oil inflammed mice at different time intervals (means +- SD)

###Percentage of injected dose per gram of tissue weight

###(n = 3/time, interval, iv)1

###Organs

###Live Staphylococcus aureus###Heat Killed Staphylococcus aureus###Turpentine oil

###30 min###1h###4h###24h###30 min###1h###4h###24h###30 min###1h###4h###24h

###Lungs###3.74+-0.6###3.66+-0.8###2.99+-0.1###1.57+-0.2###1.74+-0.8###2.04+-0.5###3.01+-0.1###0.05+-0.01###1.01+-0.06###2.96+-0.5###3.97+-0.2###1.99+-0.01

###Kidney###26.85+-1.09 29.4+-0.2###16.4+-1.0###7.16+-0.1 20.85+-1.01 19.4+-0.8###17.1+-1.01###4.05+-0.2###28.05+-1.9###18.0+-0.09###13.48+-1.9###6.06+-0.09

###Liver###5.41+-0.05 11.9+-0.9###9.66+-0.19 4.35+-0.5###5.17+-0.01 9.9+-0.06###9.52+-0.92 5.09+-0.01###5.09+-0.08 14.9+-0.56###7.30+-0.10###4.02+-0.1

###Stomach###2.73+-0.9###3.28+-0.6###3.84+-0.12 0.19+-0.7###1.73+-0.2###2.08+-0.2###4.8+-0.09###0.95+-0.07###0.70+-0.05###1.67+-0.43###3.38+-0.35###0.76+-0.1

###Intestine###2.54+-1.01 4.53+-1.2###6.99+-2.3###2.57+-0.9###1.54+-1.09 2.53+-1.0###8.99+-2.3###3.00+-0.19###4.08+-1.1###3.05+-1.02###6.22+-1.20###2.03+-0.02

###Spleen###3.93+-0.5###3.32+-0.1###1.57+-0.9###1.59+-0.2###1.93+-0.2###2.97+-0.1###1.57+-0.03 0.99+-0.09###3.03+-0.05###3.91+-0.04###0.60+-0.03###2.50+-0.07

###Blood###1.76+-0.1###3.88+-0.6###5.72+-0.25 0.32+-0.9###1.29+-0.1###2.0+-0.05###6.72+-0.05 1.50+-0.1###1.09+-0.01###2.44+-0.7###6.01+-0.09###0.06+-0.01

###Brain###0.1+-0.01###0.02+-0.1###0.03+-0.9###0.05+-0.4###0.5+-0.05###0.09+-0.4###1.03+-0.01 0.01+-0.1###0.50+-0.1###1.01+-0.5###0.06+-0.01###0.90+-0.2

###Carcass###5.3+-0.5###8.07+-1.0###4.7+-0.5###3.55+-0.8###3.3+-0.3###9.01+-0.5###3.03+-0.51 3.51+-0.01###2.95+-0.05###4.77+-1.0###4.99+-0.5###4.73+-0.1

###Bladder###1.35+-0.8###5.08+-1.9###6.91+-1.09 4.07+-0.8###1.20+-0.9###3.08+-1.0###5.91+-1. 9 4.99+-0.02###1.01+-0.04###4.05+-1.9###6.05+-1.0###3.55+-0.6

###Heart###0.54+-0.2###0.17+-0.3###0.11+-0.5###0.1+-0.01###0.14+-0.01 0.97+-0.09 0.90+-0.1###0.67+-0. 3###1.09+-0.07###1.56+-0.1###0.19+-0.1###0.14+-0.01

###Control muscle 0.21+-0.05 0.27+-0.2###0.33+-0.02 0.11+-0.01 0.18+-0.01 0.20+-0.14 0.17+-0.04 0.12+-0.12###0.15+-0.1###0.25+-0.05###0.27+-0.09###0.09+-0.02

###Target muscle###1.04+-0.1###1.7+-0.05###1.6+-0.04###0.21+-0.01 0.44+-0.1###0.78+-0.12 0.33+-0.09 0.08+-0.01###0.25+-0.02###0.73+-0.1###0.43+-0.02###0.03+-0.01

###T/NT###4.61+-0.09 6.92+-0.5###4.84+-0.01 1.90+-0.1###2.4+-0.4###3.9+-0.09###1.94+-0.05 0.6+-0.07###1.6+-0.03###2.92+-0.04###1.59+-0.19###0.3+-0.07

Conclusion

Clarithromycin is an antibiotic that is capable of controlling bacterial growth. In present study 99mTc-clarithromycin was labeled at pH 7 using 500 ug of clarithromycin and 25ug SnCl2*2H2O as reducing agent in 30 minutes incubation time. Quality control analysis was done by ITLC, and HPLC which was as high as ~99 %. The stability of 99mTc-clarithromycin in human serum was fairly high and in vitro bacterial binding assay showed higher bacterial cellular uptake. The biodistribution and scintigraphic results confirmed that mean Target to non-target ratio in the infected muscle (live S. aureus) was higher at different time intervals than in sterile-inflamed muscle (heat-killed S. aureus and turpentine oil), which indicates that 99mTc- clarithromycin may be used as a promising bacterial infection imaging agent.

Acknowledgements

Authors are grateful to Ibrar Haider for useful discussions and help during this study.

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Author:Hina, Saira; Roohi, Samina; Rajoka, Muhammad Ibrahim; Bokhari, Tanveer Hussain; Sohaib, Muhammad; Ha
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Aug 31, 2016
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