Research Article

Received 6 May 2013, Revised 4 September 2013, Accepted 27 September 2013 Published online 21 November 2013 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3135

36

99mTc-Doxycycline hyclate: a new radiolabeledantibiotic for bacterial infection imagingDerya İlem-Özdemir,a* Makbule Asikoglu,a Hayal Ozkilic,b Ferda Yilmaz,c

Mine Hosgor-Limoncu,c and Semin Ayhand

Radiolabeled antibiotics are promising radiopharmaceuticals for the precise diagnosis and detection of infectious lesions.Doxycycline Hyclate (DOX) was chosen to investigate new 99mTc-labeled antibacterial agent. Ready to use freeze dry kits wereformulated with optimum labeling conditions. Human serum stability, sterility, and pyrogenicity of kits were estimated, andgamma scintigraphy, in vivo biodistribution, and histopathological studies with bacterial infected rats were performed.

DOX were successfully labeled by 99mTc with high radiochemical purity, and the labeled compound was stable in humanserum. Kits were sterile, pyrogen-free, and stable up to 6months.

Static images depicted rapid distribution throughout the body and high uptake in bacterial infected thigh muscle. Theuptake ratios of radiopharmaceuticals in infected thigh muscle were found above 2 up to 5h.

Five hours after injection, the rats were sacrificed, and biodistribution was determined. Samples of bacterial infectedmuscle, healthy muscle, blood, liver, spleen, lung, kidney, stomach, intestine, urine and heart were weighed, and theradioactivity wasmeasured by using a gamma counter. The%ID/g of 99mTc-DOXwas found 0.23±0.06 for infected thighmuscle.

According to the imaging, biodistribution, and histopathological studies, the promising characteristics of 99mTc-DOX makethe new radiopharmaceutical valuable to examine for future studies.

Keywords: 99m-technetium; doxycycline hyclate; radiolabeled antibiotic; infection imaging

aFaculty of Pharmacy, Department of Radiopharmacy, Ege University, Bornova,Izmir, Turkey

bFaculty of Medicine, Department of Nuclear Medicine, Ege University, Bornova,Izmir, Turkey

cFaculty of Pharmacy, Department of Pharmaceutical Microbiology, EgeUniversity, Bornova, Izmir, Turkey

dFaculty of Medicine, Department of Pathology, Celel Bayar University, Manisa,Turkey

*Correspondence to: Derya İlem-Özdemir, Faculty of Pharmacy, Department ofRadiopharmacy, Ege University, Bornova, Izmir, Turkey, Tel: +90 533 446 40 26,Fax: +90 232 388 52 58.E-mail: deryailem@gmail.com

Introduction

Many bacterial infections are caused by pathogenmicroorganisms,which are capable of adhering to host cells, invading host cellsand tissues, secreting toxins, and evading the host’s immunedefenses.1,2 In many cases, it is very important for clinicaldecision-making and treatment planning not only to determinewhether or not there is an inflammatory response.3 Timelyidentification and localization of infectious foci is a critical step inappropriate treatment of patients with, or suspected of having,infectious processes.4

The radiological imaging techniques including computedtomography, nuclear magnetic resonance, and ultrasonographycannot detect infection foci in the early phase of development.Furthermore, these techniques provide information on only apart of the body. In contrast, nuclear medicine scintigraphicimaging is an excellent non-invasive method of whole-bodyscanning, which could allow to determine infectious foci in allparts of the body on the basis of pathophysiological andpathobiological changes which occur earlier in the infectionprocess.4,5 During the past years, several radiopharmaceuticalshave been developed for infection imaging.2 Today, 67Ga-citratescintigraphy, 99mTc-nanocolloid, and radiolabeled monoclonalantigranulocyte antibodies are available for this purpose. Also,radiolabeled leukocytes are still used in the diagnosis of focalbacterial infection. But the potential drawback in radiolabelingwhite cells is that patient blood is labeled ex vivo, and theprocedure carries the risk of needle-stick injury and possibleinfection.6–8 Therefore, it is an acute need to develop newdiagnostic agents for infection imaging.9,10 A novel approach

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using bacterially binding radiolabeled antibiotics was evaluatedwithdifferent researchers. 99mTc-labeled ciprofloxacin,7,11 alafosfalin,8–11

enrofloxacin,12 gatifloxacin, cefepime,13 ceftriaxone,14levofloxacin,15

and cefotaxime16 were evaluated as an infection imaging agent.Also, Örümlü has described a different radiolabeling procedure forDOX, which includes different proportions of stannous chloride,but the researcher did not evaluate the in vivo biodistributionstudies.17 İlem-Özdemir et al. radiolabeled and investigated thebiodistribution of 99mTc-DOX in sterile inflamed rats.18,19 Accordingto the biodistribution studies, 99mTc-DOX showed greater uptakein sterile inflamed tissue, whichwas high enough to distinguish fromthe background tissue.19

DOX is a broad spectrum antibacterial tetracycline derivativewith a wide range of activity against gram-positive and

Copyright © 2013 John Wiley & Sons, Ltd.

D. İlem-Özdemir et al.

gram-negative bacteria, chlamydiae, rickettsiae, and mycoplasmas.The slight chemical variation from tetracycline does not significantlyalter the bacteriostatic properties of DOX but does increase itslipophilicity, resulting in a large volume of distribution, increasedbinding to plasma proteins, and a longer elimination half-life. Thebacteriostatic effect of DOX occurs by inhibiting the bacterialprotein synthesis due to the disruption of messenger and transferRNA at the ribosomal sites.20–22

In this study, we prepared DOX cold kit for radiolabeling withtechnetium-99m (99mTc). 99mTc-DOX radiopharmaceutic stability inhuman serum, sterility, and pyrogenicity of the radiopharmaceuticalwere examined; gamma scintigraphy and biodistribution withinfected rats were investigated.

Materials and methods

All chemicals and solvents were used without furtherpurification. DOX was obtained from AppliChem (Germany).Stannous tartrate was purchased from Sigma-Aldrich (USA),and ascorbic acid was purchased from Sigma-Aldrich (UK).99mTc was eluted from the molybdenum-99 (99Mo)/99mTc-generator from Eczacıbaşı Monrol (Turkey) in the NuclearMedicine Department of Ege University. Thioglycollate brothand Tryptic soy broth mediums and all solvents were obtainedfrom Merck (Germany). Dose Calibrator (Atomlab 100, BiodexMedical Systems) was used for counting radioactive samples.Escherichia Coli bacteria were obtained from PharmaceuticalMicrobiology Department of Ege University. The Animal EthicsCommittee of the Ege University gave approval for the animalexperiments (Number: 2010–37, 2010). Results are reported asmean± standard error.

Preparation of 99mTc-DOX

99mTc-DOX was prepared from lyophilized cold kit, whichpreviously prescribed by İlem-Özdemir et al.18 Briefly, the kitincluded 1mg DOX, 30μg stannous tartrate, and 0.1mg ascorbicacid. One millimeter of 0.9% sodium chloride was injected in tothe kit to dissolve the dried powder. Then the content wasradiolabeledwith (370MBq) 99m-technetium sodiumpertechnetate(99mTcO4

- ). The vial was shaken and incubated at roomtemperature for 15min. After incubation, the reaction productwas filtered through a 0.22-μm pore size filter for sterilization.

Radiochemical analysis of 99mTc-DOX

The radiochemical purity was determined by instant thin layerchromatography (ITLC) and radio high performance liquidchromatography (R-HPLC) studies, which were previouslydescribed.18

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Instant thin layer chromatography analysis

One drop of reaction product was spotted on two ITLC-silica gelchromatographic paper strips (each of 1 × 8 cm). The first stripwas developed in acetone, whereas the other one wasdeveloped in acetonitrile/water/trifluoroacetic acid (50/25/1.5,respectively) solvent system. After completing the development,both strips were dried and scanned in a TLC scanner (Bioscan AR2000), and percentages of reduced/hydrolyzed 99mTc, free 99mTc,and 99mTc-DOX were calculated.

Copyright © 2013 JohJ. Label Compd. Radiopharm 2014, 57 36–41

Radio high performance liquid chromatography analysis

99mTc-DOX radiochemical purity was analyzed by an Ultra-HPLCsystem equipped with a C18 column connected to a photodiodearray detector and additional thallium doped sodium iodide (NaI(Tl)) gamma detector for the 99mTc compounds. Chromatographicanalysis was performed with a 10-μL reaction mixture. The flowrate was 0.5mL/min for analytical runs. In all runs, the eluent was0.1% trifluoroacetic acid in acetonitrile.

Stability of 99mTc-DOX in human serum

To test the serum stability of 99mTc-DOX, 100-μL reactionmedium was added to 900μL of freshly prepared human serum.The mixture was incubated at 37°C for 24 h. Two drops of samplewas spotted on chromatographic strips at different time intervalsup to 24 h, and ITLC studies were performed to determine thestability of 99mTc-DOX in human serum.

Microbiological analysis of 99mTc-DOX kit

Sterility test

The sterilities of the kits were tested by direct inoculation methodaccording to the European Pharmacopeae 6.0 recommendations.The dilutions of the kits were aseptically inoculated to thesterilized Thioglycollate broth and Tryptic soy broth medium vialsand incubated at 35 ± 2°C. At the end of the incubation, absenceof clearly visible growth of microorganisms in the vials confirmsthe sterility.

Pyrogenicity test

Pyrogenicity test was performed by the gel clot method, which isbased upon the reaction between bacterial endotoxin and thespecific lysate. In the presence of endotoxin, the gel is formed viaa clotting reaction, and the sample failed. Endotoxin limit valueof the kit and themaximum valid dilutionwas estimated accordingto European Pharmacopeia 6.0. The Limulus amebocyte lysate testis performed by Pyrotell (Associates of Cape Cod, Incorporated,Falmouth, Massachusetts) gel-clot formulation.

Animal studies

Wistar albino rats (200–250 g) were used for animal studies.Experiments with rats were performed according to a protocolapproved by Animal Ethics Committee of the Ege University.

Induction of infection model

E. coli suspension was used to create infection model (23).Groups of six rats were intramuscularly injected with 200μL(4 × 1010 cfu/1mL) E. coli suspension into the right thigh muscle.Then the infection was allowed to develop for 24 h.

Scintigraphic imaging studies of 99mTc-DOX

After the infection focus was allowed to develop for 24 h,swelling appeared, and gamma scintigraphy studies wereperformed. Rats were intravenously injected with 99mTc-DOX(3.7MBq) via tail vein, and during the scintigraphy studiesanimals were under anesthetize with Ketamine/Xylazin cocktail.The scintigraphic images were obtained with a dual headgamma camera (Infinia General Electric) equipped with a low-energy, high-resolution collimator viewing the whole body ofrats. After administration of radiopharmaceuticals, serial staticimages were acquired in a 256× 256 matrix for 60 s each, atdifferent time intervals (5min, 1, 2, 3, and 5 h post-injection).

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Biodistribution studies

Five hours after injection, the rats were sacrificed, andbiodistribution was determined. Samples of infected muscle,healthy muscle, blood, liver, spleen, lung, kidney, stomach,intestine, urine, and heart were weighed, and the radioactivitywas measured using a gamma counter (Sesa Uniscaller). Theresults were expressed as the percentage of injected dose pergram of tissue (%ID/g).

Histopathology

After the time of sacrifice, the infected and healthy rat musclespecimens dissected out and were fixed with 10% neutral bufferformalin and embedded in paraffin using routine procedures.Sections of 4 to 5μm in thickness were stained withhematoxylin/eosin for histological examination. All biopsyspecimens were interpreted in a blinded fashion by conventionallight microscopy.

Statistical analysis

The calculation of means and standard deviations were made onMicrosoft Excel. One-way analysis of variance was used todetermine statistical significance. Differences at the 95%confidence level (p< 0.05) were considered significant.

Results

Radiolabeling and radiochemical purity

An instant cold kit was prepared and contained DOX, stannoustartrate, and ascorbic acid. The radiochemical purity of 99mTc-DOXwas greater than 90%, acquired via both ITLC and also R-HPLCstudies. Over a period, the resulting complex was quite stable,and radiochemical purity was found ˃90% up to 6 h.

Figure 1. Stability of99m

Tc-DOX in human serum.

Figure 2. Scintigrams of infected rat’s right thigh muscle up to 5 h after injection (T

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Stability of 99mTc-DOX in human serum

During incubation in human serum, compound was stable asdetermined by ITLC. As shown in Figure 1, the percentage of99mTc-DOX was decreased from 97.46 ± 0.62 to 94.02 ± 0.41within 24 h.

Microbiological analysis of 99mTc-DOX kit

According to sterility test, because there was no growth inbatches, kits were sterile. Also, gel clot test showed that kits werepyrogen-free.

Scintigraphic imaging studies of 99mTc-DOX

The uptake of 99mTc-DOX following intravenous (IV) administrationswere assessed on static images. The images depicted rapiddistribution throughout the body and uptake in the infectedthigh muscle within 1 h after injection. As can be seen in Figure 2,there was a higher activity of infected thigh muscle as comparedwith healthy thigh muscle.

For quantitative evaluation, regions of interest were drawnaround the target (infected thigh muscle) and non-target(healthy thigh muscle) regions of the rats. The 99mTc-DOX uptakewas calculated by dividing the average counts per pixel withinthe region of target to the average counts per pixel within theregion of non-target.

According to the imaging studies, the uptake ratios of 99mTc-DOX (target/non-target) were found 2.13 ± 0.56, 2.23 ± 0.55, and2.24 ± 0.84 at 5min, 3, and 5 h after injection, respectively(Table 1). These ratios indicated a significant uptake of 99mTc-DOX in infection foci. The data suggest that 99mTc-DOX remainedat the infection foci during the whole experiments, there beingno statistically significant differences in the ratios during thestudied period (p≥ 0.05).

Biodistribution studies

On the basis of data presented in Table 2, biodistribution of99mTc-DOX was determined for lung, kidney, spleen, urine,intestine, heart, blood, liver and stomach infected and healthythigh muscle.

99mTc-DOX was mainly excreted by renally in rats. Stomachuptake of 1.44 ± 0.26 %ID/g was high, but because the liveruptake is very low (0.20 ± 0.05), these results did not exhibitedthe significant amount decomposition of the compound.99mTc-DOX was found to accumulate in the infected thighmuscle of rats to the 0.23 ± 0.06 %ID/g.

= target, NT = non-target).

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Table 1. Target/non-target ratios from ROI analysis 99mTc-DOK at different time intervals

Time after injection (h) Target/non-target

0 2.13 ± 0.561 2.15 ± 0.392 2.10 ± 1.213 2.23 ± 0.555 2.24 ± 0.84

Values expressed as the mean± standard deviation, n= 6

Table 2. Biodistribution of 99mTc-DOX in infected rats 5 hpost-injection (%ID/g)

Organ %ID/g Organ %ID/g

Lung 0.13 ±0.03 Heart 0.09 ± 0.02Kidney 6.51 ± 2.49 Blood 0.23 ± 0.02Spleen 0.11 ± 0.01 Liver 0.20 ± 0.05Urine 16.48 ± 0.94 Stomach 1.44 ± 0.26SmallIntestine

2.20 ± 1.57 Infected muscle 0.23 ± 0.06

LargeIntestine

0.15 ± 0.12 Healthy muscle 0.09 ± 0.04

Values expressed as the mean± standard deviation, n= 6

D. İlem-Özdemir et al.

According to the biodistribution studies, target/non-target ratio of the 99mTc-DOX was found as 2.62 ± 0.88, 5 hafter injection.

Figure 3. (A) Healthy rat muscle and (B) infected rat muscle microscopic appearanc

Copyright © 2013 JohJ. Label Compd. Radiopharm 2014, 57 36–41

Histopathology

Conventional haematoxylin and eosin-stained sections (microscopicappearances) of the infected and healthy rat muscle tissue areshown in Figure 3. Infected rat muscle exhibits acute suppurativeinflammation with polymorphonuclear leucocytes infiltration,which indicates the presence of bacterial infection.

Discussion

In this study, DOX was preferred to label with 99mTc because ofits wide range of activity against gram-positive and gram-negative bacteria, and suitable chemical properties for labelingprocess. DOX was successfully labeled with 99mTc from an instantcold kit without any incubation and purification. Radiochemicalpurity of 99mTc-DOX was ˃90%. According to in vitro stabilitystudies, the labeled compound was stable for a period of up to6 h. In addition, 99mTc-DOX complex was stable in human serumduring incubation period up to 24 h.

Motaleb was radiolabeled lemofloxacin and ofloxacin with99mTc. According to radiochemical purity studies, the rate offormation of 99mTc-lomefloxacin started relatively slowly with ayield of 83.8% at 1min. The highest yield of 93.6% was achievedat 30min. The complex was stable up to 120min; after that, theyield decreased again (>80%). Although 99mTc-ofloxacin wasformed at once on the addition of 99mTc to the reaction mixture(96.6%), the formed complex remained only stable up to120min; after that, the yield decreased again (>80%). Accordingly,the radiochemical purity and stability of 99mTc-DOX were higherthan 99mTc-lomefloxacin and 99mTc-ofloxacin for up to 24h (24).

Roohi et al. described the labeling of kanamycin with 99mTc.Radiochemical purity, stability, and in vivo biodistribution ininfected rats and rabbits were evaluated. Accordingly, thecomplexation of 99mTc with kanamycin was not rapid, andmaximum labeling efficiency was achieved after 30min. The

e viewed by ×4 and ×10 microscopy.

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resulting complex of 99mTc-kanamycin was quite stable, andlabeling of ≥98% is maintained for up to 6 h. When thepreparation was incubated with normal human serum at 37°C,the total impurities were found less than 5% (25).

The rat infection model resulted in a focal swelling and fewerin thigh muscles and validated by the pathological evidence.99mTc-DOX was rapidly distributed after IV injection as shownby the renal elimination. Since the soft tissue uptake was low,infectious site was easily captured. The rapid uptake at the siteof infection was seen on images at 1 h after injection. Accordingto gamma scintigraphy studies, rats with infection foci injectedwith 99mTc-DOX showed a target/non-target ratio as 2.24 ± 0.84,5 h after injection.

Table 2 also presents the infected thigh and healthy thighradioactivity obtained at 5 h after administration of 99mTc-DOX.According to biodistribution studies, the target/non-target thighmuscle ratio indicated that higher uptake in infectioninduced muscle (>2.50).

The tissue distribution of 99mTc-kanamycin was examined withrats, which were infected by S. aureus. The 99mTc-kanamycin wasrapidly distributed after i.v. administration. 0.5, 4, and 24 h afterinjection the target thigh/healthy thigh radioactivity ratio wasfound to be 2.5, 2.35, and 2.46, respectively. Although ease ofpreparation, good stability, and animal experiments has shownthat 99mTc-kanamycin localizes in bacteria infected sitessignificantly, the radiopharmaceutic showed high accumulationin liver (22.25% ID/g). Accordingly, the liver uptake of 99mTc-DOXwas significantly lower (0.20% ID/g) than 99mTc-kanamycin (25).

Akhtar et al. investigated the 99mTc-labeled antimicrobialpeptide ubiquicidin accumulates in E. coli and S. aureus infection,and the labeled peptide was showed less uptake in E. coli thanS. aureus. These results indicate that the different bacterial-induced infection models cause differences between the uptakeratios of radiopharmaceutics (23).

99mTc-lomefloxacin and 99mTc-ofloxacin could be used fordetecting infection sites without any purification within 2 h afterpreparation. 99mTc-lomefloxacin was accumulated at the site ofinfection with higher than 99mTc-ofloxacin. 99mTc-lomefloxacinshowed higher yield and better biodistribution properties than99mTc-ciprofloxacin. The rats with infectious lesions injected with99mTc-lomefloxacin showed a target/non-target muscle ratioequal to 6.5 ± 0.5, whereas target/non-target for 99mTc-ofloxacinwas 4.3 ± 0.6. Both 99mTc-fluoroquinolones showed greateruptake in infected tissue than 99mTc-ciprofloxacin (target/non-target = 3.8 ± 0.8). Instead of these good utilities, theradiopharmaceuticals could not be used in nuclear medicinedepartments because of their stability problems (25).

İlem-Özdemir et al. previously published a paper about thebiodistribution of 99mTc-DOX in sterile inflamed rats.19 Sterileinflamed thigh muscle model was induced with direct injectionof turpentine oil in the left thigh muscle of rats. The right thighmuscle was used as control. Accumulation of the tracer ininflamed areas was assessed by gamma scintigraphy studies atdifferent time intervals after the injections (0, 1, 2, 3, 4, and5 h). For quantitative evaluation, regions of interest were drawnaround the target (inflamed thigh muscle) and non-target(healthy thigh muscle) regions of the rats. The 99mTc-DOX uptakewas calculated by dividing the average counts per pixel withinthe region of target to the average counts per pixel within theregion of non-target. According to gamma scintigraphy studies,target/non-target ratios was found to be 2.57 ± 1.54, 3.18 ± 1.26,4.55 ± 2.61, 3.69 ± 2.24, and 3.64 ± 2.60 for 0, 1, 2, 3, 4, and 5 h

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after injection, respectively.19 These ratios indicated a significantuptake of 99mTc-DOX in both bacterial infected and sterileinflamed thigh muscle.

Conclusion

DOX was radiolabeled with high radiochemical purity. Thelabeled compound was found to be stable in human serum upto 24 h. The bacterial infection imaging capacity of 99mTc-DOXwas evaluated. On the basis of the gamma scintigraphy andbiodistribution studies, 99mTc-DOX has higher uptake ininfected thigh muscle than non-infected muscle. According toprevious studies, 99mTc-DOX also has higher uptake ininflamed thigh muscle than healthy thigh muscle.19 In vitro andin vivo characteristics of the 99mTc-DOX are promising forinflammatory process imaging, but the radiopharmaceuticalcannot be able to distinguish E. coli-induced bacterial infectionfrom sterile inflammation.

Acknowledgements

This study was supported by The Scientific and TechnologicalResearch Council of Turkey (Tubitak-110 S 229). The authorswould like to acknowledge the support of T.R. Prime MinistryState Planning Organization Foundation Grant Project Number:09DPT001 for TLC Scanner. Also, the authors thanks EgeUniversity Nuclear Medicine Department for their assistance forthe experiments.

Conflict of Interest

The authors did not report any conflict of interest.

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