Scholarly article on topic 'Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection'

Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection Academic research paper on "Chemical sciences"

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{Ceftriaxone / Technetium-99m / "Infection imaging" / SPECT}

Abstract of research paper on Chemical sciences, author of scientific article — Ankur Kaul, Puja P. Hazari, Harish Rawat, Baljinder Singh, Tek C. Kalawat, et al.

Summary Objective In this study we sought to assess the efficacy of a technetium-99m (Tc-99m)-labeled third-generation cephalosporin as an infection imaging agent in the accurate detection of the sites of bacterial infection in vivo. Design Ceftriaxone (CRO) was formulated into a ready-to-use single-vial cold kit with a shelf-life of over 6 months and was successfully labeled with technetium. The radiolabeled drug, Tc-99m-CRO, was subjected to the following preclinical evaluations: radiochemical purity, in vitro and in vivo stability, bacterial binding assay, and pharmacokinetic studies in animals and in human patients. Results The kit formulation exhibited excellent radiolabeling efficiency (∼99%) and high in vitro and in vivo stability. The radiolabeled drug exhibited slow blood clearance (12% at 4h), and the high protein binding and excretion pattern of the labeled formulation mimics the reported pharmacokinetic profile of the drug alone. In the animal model, scintigraphy scans showed higher uptake of the radiopharmaceutical in infectious lesions, even at 1h post-administration, in comparison to inflammatory lesions. The clinical evaluation of Tc-99m-labeled CRO showed a diagnostic accuracy of 83.3%, and a sensitivity and specificity of 85.2% and 77.8%, respectively. Conclusions This kit formulation has the potential for imaging bacterial infections with much higher sensitivity and specificity as compared to other Tc-99m-labeled antibiotics available as convenient ready-to-use kits in routine clinical practice.

Academic research paper on topic "Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection"

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Preliminary evaluation of technetium-99m-labeled ceftriaxone: infection imaging agent for the clinical diagnosis of orthopedic infection

Ankur Kaula, Puja P. Hazaria, Harish Rawata, Baljinder Singh b, Tek C. Kalawatc, Sarika Sharmab, Anil K. Babbar3'*, Anil K. Mishra^*

a Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. SK Mazumdar Road, Near Timarpur, Delhi, 110054, India b Department of Nuclear Medicine, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India c Department of Nuclear Medicine, Sri Venkateshwara Institute of Medical Sciences (SVIMS), Tirupati, India

SUMMARY

Objective: In this study we sought to assess the efficacy of a technetium-99m (Tc-99m)-labeled third-generation cephalosporin as an infection imaging agent in the accurate detection of the sites of bacterial infection in vivo.

Design: Ceftriaxone (CRO) was formulated into a ready-to-use single-vial cold kit with a shelf-life of over 6 months and was successfully labeled with technetium. The radiolabeled drug, Tc-99m-CRO, was subjected to the following preclinical evaluations: radiochemical purity' in vitro and in vivo stability' bacterial binding assay, and pharmacokinetic studies in animals and in human patients. Results: The kit formulation exhibited excellent radiolabeling efficiency (~99%) and high in vitro and in vivo stability. The radiolabeled drug exhibited slow blood clearance (12% at 4 h), and the high protein binding and excretion pattern of the labeled formulation mimics the reported pharmacokinetic profile of the drug alone. In the animal model, scintigraphy scans showed higher uptake of the radiopharmaceutical in infectious lesions, even at 1 h post-administration, in comparison to inflammatory lesions. The clinical evaluation of Tc-99m-labeled CRO showed a diagnostic accuracy of 83.3%, and a sensitivity and specificity of 85.2% and 77.8%, respectively.

Conclusions: This kit formulation has the potential for imaging bacterial infections with much higher sensitivity and specificity as compared to other Tc-99m-labeled antibiotics available as convenient ready-to-use kits in routine clinical practice.

© 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

ARTICLE INFO

Article history:

Received 19 January 2012

Received in revised form 26 July 2012

Accepted 25 October 2012

Corresponding Editor: J. Peter Donnelly,

Nijmegen, the Netherlands

Keywords: Ceftriaxone Technetium-99m Infection imaging SPECT

1. Introduction

The detection of any infection by nuclear medicine techniques relies on the physiological and biochemical changes that occur at the site of the lesion, which appear much earlier than anatomical changes.1-3 Single photon emission computed tomography (SPECT), a nuclear medicine technique that can be used in clinics, gives a more detailed localization of the lesion and can provide crucial information, especially in patients with osteomyelitis and those suspected of deep-seated infections of joint prostheses or implants. The most widely used radioisotope for this technique is technetium-99m (Tc-99m) due to its favorable physical properties; the 140 keV energy of gamma emission and half-life of 6 h offers ideal nuclear medicine imaging properties. Therefore a Tc-99m radioisotope tagged to a biomolecule or small organic

* Corresponding authors. Tel.: +91 11 23905117; fax: +91 11 23919509. E-mail address: akmishra63@gmail.com (A.K. Mishra).

molecule of interest has the potential to make an ideal radiopharmaceutical for diagnosis.4

Most radiopharmaceuticals reported in the literature for the diagnosis of infection, such as radiolabeled polyclonal or monoclonal immunoglobulins, cytokines, peptides, and liposomes, are in fact only tracers of the inflammation process.5-7 Certain radiopharmaceuticals that specifically bind to a variety of bacteria, such as radiolabeled antibiotics, have been shown to detect and locate infection, but have not shown a high specificity to discriminate between bacterial infection and sterile inflammation.8-14 The first such radiopharmaceutical proposed was a two-vial kit of ciprofloxacin tagged with Tc-99m (Infecton®), which was further modified to a single-vial instant kit. However, bottlenecks in the use of the kit such as the need for filtration during reconstitution caused additional radiation burden and led to the loss of

radioactivity.15-17

Looking at the need for a better infection imaging formulation with higher specificity towards bacteria, we labeled ceftriaxone (CRO) (Figure 1a), a third-generation

1201-9712/$36.00 - see front matter © 2012 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijid.2012.10.011

cephalosporin. As a cell peptidoglycan inhibitor, CRO has a high therapeutic index because this basic unit of the cell wall structure is not found in eukaryotic cells. The unique mechanism of action of cephalosporins in which the penicillin disrupts the ratio between penicillin-binding protein (PBP)-mediated peptidoglycan synthesis and murein hydrolase activity, results in autolysis of the cell wall structure. CRO is a broad-spectrum semi-synthetic cephalosporin with a long half-life and in vitro activity against staphylococci and Gramnegative aerobic bacilli except for Pseudomonas.18 Structure-activity relationship (SAR) studies of CRO show that the presence of the 3-thiotriazine ring augments metabolic stability and protein binding, thereby increasing its circulation time, while its 7-aminothiadiazole alkoxy imine group ensures extended beta-lactamase stability and enhanced anti-staphy-lococcal activity.19

A current biomedical challenge is the detection and identification of orthopedic bacterial infections, because clinical cases that are detected by culture positivity may only represent a small fraction of infecting microbes, and identifying the causative species and its sensitivity to a particular antibiotic is critical for effective antimicrobial therapy. Therefore, any diagnostic test will need to be proven useful and cost-effective in everyday clinical practice by virtue of its sensitivity and specificity.

The objective of the present work was the development of a biologically active Tc-99m-CRO single-vial kit formulation, which can be easily reconstituted, to identify active septic foci in the experimental model of Staphylococcus aureus-induced infectious lesions in the rabbit, with further application in clinics after approval from the human ethics committee.

2. Methods

2.1. Chemicals

Ceftriaxone was procured from Ranbaxy Industries Ltd and Tc-99m-pertechnetate was supplied by BRIT, Regional Center, Delhi. Stannous tartrate and gentisic acid were procured from Sigma Aldrich. All other chemicals used were obtained from major suppliers. A Capintec Caprac-R® gamma scintillation well-type counter was used for the determination of activity. The Infinia Hawkeye® GE gamma scintillation camera was used for imaging of animals. Clinical patient imaging was done using Siemens Biograph® and E.Cam® systems.

2.2. Radiolabeling with Tc-99m using a ready-to-use single-vial cold kit of ceftriaxone

A lyophilized single-vial kit containing 2.5 mg of the sodium salt of CRO, 200 mg of stannous tartrate, and 200 mg of gentisic acid at appropriate pH was developed in-house. The kit was reconstituted by adding 1-2 ml of sterile Tc-99m-pertechnetate (74740 MBq) to the lyophilized vial with continuous mixing. The reaction mixture was incubated at room temperature for 30 min.

2.3. Radiochemical purity

The radiochemical purity of Tc-99m-CRO was assessed by ascending instant thin-layer chromatography (ITLC) using silica gel-coated fiber glass sheets (Pall Corporation) and two solvent systems, namely (1) 100% acetone and (2) a solvent mixture of ethanol, ammonia, and water (2:1:5 v/v), as mobile phases. The radioactive contaminants were identified as reduced/hydrolyzed (R/H) Tc-99m (Rf = 0.0) and free Tc-99m-pertechnetate (Rf = 1.0) and the labeled product remained at the point of application when 100% acetone alone was used as the mobile phase. The labeled

products moved with the solvent front (Rf =1.0) along with free Tc-99m-pertechnetate, leaving R/H Tc-99m at the origin (Rf = 0.0) when the solvent mixture was used as mobile phase. This was further confirmed by chromatographic analysis by scanning the developed TLC plate in an EZ TLC scanner.

2.4. Lyophilization and stability of the ceftriaxone kit formulation

The samples were subjected to lyophilization using a Triad laboratory freeze-dryer. Pre-freezing of the kit was carried out at a temperature of -40 °C for 7.5 h. The temperature was subsequently increased to -10 °C at 0.6 °C/min for primary drying for 5 h. Further ramping of the temperature by 0.25 °C/min was done with the temperature increasing to 20 °C and the chamber pressure maintained at 0.03 mbar for 15 h. The stability of the ready-to-use kit (n = 6) was determined at 7, 14, 30, 60, 90, and 180 days after storage under the following conditions: (1) in a freezer at -20 °C with and without nitrogen atmosphere, (2) in a refrigerator at 4 °C, and (3) at room temperature (25 °C).

2.5. In vitro stability study

The radiolabels of the drug were tested for in vitro stability by ascending ITLC. For in vitro stability in physiological saline and serum, 100 ml of the radiolabel was mixed with 2 ml each of 0.9% saline and serum. ITLC was carried out to assess the efficiency of labeling after incubation at 37 °C for 30 min.

2.6. Microorganisms

Overnight cultures of S. aureus bacteria were prepared on tryptone soya agar (Oxoid) plates at 32 °C. Aliquots of suspensions of harvested microorganisms containing 2 x 108 colony-forming units (CFU) were kept in 1 ml of buffered peptone water (Oxoid) at 4 °C for a maximum of 1 week.

2.7. Bacterial binding assay

Bacterial binding of Tc-99m-CRO was assessed with both living and heat-killed S. aureus bacteria. Four groups of bacteria were defined and used in sets of six vials: group A was the blank control group, groups B and D consisted of live bacteria, and group C consisted of heat-killed bacteria. The radiotracer for the study was prepared with a final concentration of 30 mg/ml of CRO and 600 mg/ml of diethylenetriamine pentaacetate (DTPA).

A bacterial suspension (0.9 ml) containing approximately 108 CFU was taken, and exactly 0.1 ml of phosphate-buffered saline (PBS) containing approximately 5 MBq of radiolabel was added to the test tube. The mixture was incubated for 1 h at 37 °C and then centrifuged for 5 min at 2000 rpm at a temperature of 4 °C; the pellet was then resuspended in 1 ml ice-cooled PBS. The resuspended pellet was then centrifuged again for 5 min, the supernatant separated and 1 ml of ice-cooled PBS added.

The supernatant was removed and the activity in the bacterial pellet was determined using the gamma counter. The radioactivity in the bacterial pellet was expressed as the percentage of the Tc-99m activity bound to bacteria with regard to total Tc-99m activity.

2.8. Statistical analyses

All mean values were expressed as the percentage of the injected dose per gram or ratio ± standard deviation (SD). A statistical analysis was performed of the clinical outcomes from the two imaging centers using the Student's t-test.

Figure 1. Chemdraw1 structures of (a) ceftriaxone and (b) proposed binding of ceftriaxone with Tc-99m.

2.9. Animal studies

2.11. In vitro and in vivo protein binding study

All animal experiments were carried out in accordance with the guidelines of the INMAS Animal Ethics Committee. Mice weighing 15-25 g of BALB/c strain were used for the biodistribution studies. New Zealand albino rabbits weighing 2.5-3.0 kg were used for the blood clearance studies, serum protein binding study, and gamma scintigraphy. The animals were taken at random from the stock colony and used from the experimental animal facility at the institute. The animals were fed laboratory chow pellets and had free access to food and water. The animals were in a room with light during the daytime and no light from 19:00 h in the evening until morning; the temperature was maintained at approximately 25 °C.

2.10. Blood clearance study

Tc-99m-CRO (74 MBq/0.1 ml) was administered to rabbits (n = 3) through the ear vein, and blood samples were collected at different time intervals post-administration. The radioactivity in blood samples was measured in a well-type gamma counter and was calculated as the percentage of the injected dose.

For the in vitro protein binding study, 37 MBq of Tc-99m-CRO in 0.1 ml was mixed with 2 ml of plasma (n = 6). Aliquots were taken at various time intervals and proteins were precipitated by adding equal volumes of 12.5% trichloroacetic acid (TCA) and plasma. The radioactivity in the precipitate and supernatant was measured in a well-type gamma counter. The plasma protein binding was expressed as the fraction of total activity in the sample. For in vivo studies, 74 MBq of radiotracer was administered to the rabbit and blood samples were withdrawn at various time intervals. The plasma was separated from the samples and was processed in the same way as described above for the in vitro studies.

2.12. Biodistribution study in normal mice

The in vivo distribution of Tc-99m-CRO was studied in 2-month old BALB/c mice (n = 3; each weighing approximately 22 g). An aliquot of 100 ml of Tc-99m-CRO (3.7 MBq) was administered intravenously (IV) to each mouse through the tail vein. The animals were sacrificed at different time intervals and the different organs were removed and collected into pre-weighed tubes. The

radioactivity in each organ was counted using a well-type gamma spectrometer and count per min (CPM) values were decay-corrected; results were calculated as the percentage injected dose per gram of wet tissue. All animal studies were approved by the local experimental animal ethics committee and performed in accordance with their guidelines.

2.13. Experimental animal models

Induction of sterile inflammation: 100 ml of sterile turpentine oil was injected deep into the left forearm of New Zealand albino white rabbits (n = 3)2 days before scintigraphy studies.

Induction of bacterial infection: a bacterial suspension of S. aureus (concentration 107 CFU/100 ml) was injected deep into the left forearm of New Zealand albino white rabbits (n = 3) 24 h before the imaging study.

2.14. Experimental animal model scintigraphy

Scintigraphy in rabbits was carried out after IV administration of 0.1 ml of Tc-99m-CRO (74 MBq) through the ear vein. The animals were anesthetized by intramuscular injection of ketamine hydrochloride (50 mg/kg body weight) 10 min before imaging. The animal was fixed on a board in the posterior anterior position and imaging was performed using a planar gamma camera (GE Hawkeye®) at 1, 4, and 24 h post-administration. Regions of interest (ROIs) were drawn around the infectious/inflammatory focus and over the contralateral control zone for comparison, and analyzed using Entegra® software (Elgems, Haifa, Israel).

2.15. Preliminary clinical studies

All clinical studies were carried out in accordance with the guidelines of the institutional ethics committee. Patients who were on any antibiotic treatment, who had undergone a recent amputation/debridement, or who had a documented hypersensi-tivity to cephalosporins were excluded from the study. Written and informed consent was obtained from all the patients. The purpose of the study was explained to each study participant.

Thirty-six patients (22 male and 14 female; mean age 47.44 years, range 18-78 years) with a clinical and radiological suspicion of an orthopedic infection were recruited. All the patients underwent a 3-phase bone scan (740 MBq, Tc-99m-methylene diphosphonate (MDP), IV injection) followed 4 days later by Tc-99m-CRO scanning (static/isotime images at 1, 4, and 24 h after IV administration of 555.0 MBq activity of the radiotracer). Whole body Tc-99m-CRO scanning was also done to study the biodistribution of the radiotracer. Sample aspirates from the site involved were taken and analyzed for microbial growth. A representative case, an 18-year-old male patient, presented with pain, swelling, and a non-healing ulcer with active pus discharge on the right leg (distal part medially) of 2-month duration; microbial culture of a wound swab grew S. aureus.

3. Results

600000 500000 ■ 400000 -I 300000 ■ 2D0000 -100000 ■

0 5 10 15 20

Rf(cm)

Figure 2. Radiochromatogram profile of Tc-99m-labeled ceftriaxone.

showed the Rf of the labeled CRO to be 0.9, while free technetium was found along with the solvent front (Figure 2). Even after 24 h of storing the preparation at room temperature, more than 95% of radioactivity was found to be coupled with CRO. Since the radiolabeled drug remains in the complexed form (above 90%) up to 24 h, it appears to be a suitable candidate for diagnostic imaging. Also, considering the decay factor and the biological halflife of 8 h during which almost all the bound complex is excreted from the body, it will not cause any additional radiation burden to the patient.

On incubation with normal saline and rabbit serum, the radiolabel was found to be stable up to 24 h. However, at 24 h the in vitro stability was reduced, as 5-6% free Tc-99m-pertechnetate was found in the preparation (Table 1).

Lyophilization of the kit was efficiently and successfully carried out using our protocol, and a fluffy off-whitish lyophilized powder was obtained. Storage of the kit was studied in three different temperature conditions. The shelf-life of the refrigerated lyophi-lized kit was found to be more than 6 months and the results showed no significant differences in stability of the kit at 4 °C and at -20 °C, while there was a difference for storage at room temperature. Hence, for optimal labeling efficiency, the formulated kit should be stored in a refrigerator at 4 °C.

Following S. aureus incubation at 37 °C for the bacterial binding assay, results showed that the binding of labeled CRO to heat-killed bacteria and live bacteria was similar, while a significantly lower fraction of bacterial binding was found in the case of labeled DTPA as compared to the labeled drug (Table 2).

Blood clearance of Tc-99m-CRO in whole blood with time after IV administration in rabbits exhibited bi-exponential clearance with 12% of the radioactivity in blood circulation at 4 h (Figure 3). In vitro as well as in vivo protein binding studies showed that Tc-99m-CRO was highly protein-bound (92% at 4 h) (Figure 4). The biodistribution of Tc-99m-CRO in the various organs of mice at various time intervals post-administration is shown in Table 3. Among all the organs studied, the kidneys showed a maximum uptake of 12.2% of the injected dose per whole organ at 1 h, which increased to 15.8% at 4 h. Significant radioactivity in the liver,

The optimum labeling yield (~99%) was achieved when 200 mg stannous tartrate was used in the presence of gentisic acid for complexing 2.5 mg CRO with 74-740 MBq of Tc-99m-pertechne-tate. Quality control tests using ITLC revealed that Tc-99m-CRO contained less than 1% free Tc-99m-pertechnetate and 2-3% R/H Tc-99m. When the preparation was stored at room temperature, there was no significant degradation of the product up to 4 h. The proposed structure of the 1:1 complexation of Tc-99m with CRO drug is shown in Figure 1b. Radiochemical purity was further assessed through EZ TLC scanner, and the radiochromatogram

Table 1

In vitro stability of Tc-99m-ceftriaxone

Incubation time (h) % Radioactivity

Free Tc-99m R/H Tc-99m Tc-99m-CRO

0 0.8 1.8 97.4

1 1.0 1.8 97.2

2 1.1 2.1 96.8

4 1.8 2.6 95.4

24 2.6 5.8 91.4

R/H, reduced/hydrolyzed; CRO, ceftriaxone.

Table 2

Percentage bacterial binding after incubation with Tc-99m-ceftriaxone in groups B and C, and with Tc-99m-diethylenetriamine pentaacetate in group D (± SD of six vials in each group)

Group % binding

Live-bacteria (group B), Tc-99m-CRO 4.35 ± 1.16

Heat-killed bacteria (group C), Tc-99m-CRO 3.31 ± 0.86

Live-bacteria (group D), Tc-99m-DTPA 0.49 ± 0.22

CRO, ceftriaxone; DTPA, diethylenetriamine pentaacetate.

intestines, and kidney suggests its excretion through both the hepatobiliary and renal routes.

Scintigraphy studies in animal models showed that the uptake of the radiotracer was appreciably higher in the infected muscle in comparison to the inflamed muscle (Figure 5). Tc-99m-CRO showed only background activity in turpentine-induced sterile inflammation, and activity detected at the site was similar to that in the contralateral muscle area. In the case of S. aureus-induced infection, Tc-99m-CRO had a very high target to non-target ratio, which kept increasing, reaching a maximum of 4.5 at 24 h (Table 4).

In this study we also evaluated the clinical utility of Tc-99m-labeled CRO for the detection of orthopedic infections where scan findings (CRO scan) were positive in 25/36 and culture was positive in 27/36 patients (Table 5). The diagnostic accuracy of the technique was found to be 83.3%, and the sensitivity and specificity were 85.2% and 77.8%, respectively. The results presented are the cumulative data from two different imaging centers. A statistical analysis was performed using the Student's t-test and confirmed that there was no statistical difference with respect to outcome between the two centers.

The radiolabeled kit showed encouraging results in localizing orthopedic infections, especially in the setting of acute bacterial infection (Figure 6). The representative scan shows a high target to non-target ratio at 1 h post-injection in the patient with an active bacterial infectious lesion on the right leg, which was confirmed through microbial culture.

4. Discussion

The design of an ideal infection imaging radiopharmaceutical requires the optimization of several factors: (1) high specificity in vivo/target to non-target ratio, (2) non-immunogenic, (3) low toxicity, (4) cost-effective, (5) easy to prepare, and (6) widely available.20 This kit was formulated keeping in mind that the long shelf life of the kit preparation and simple convenient method to

Figure 4. In vitro and in vivo plasma protein binding of Tc-99m-ceftriaxone expressed as a fraction of total radioactivity in the sample.

Sterile Inflammation Pk A- lh Septic Lesion B- lh

C 4h 4 D. 4h

E 24h si- F- 24h

Figure 5. Whole body scintigraphy of experimental models of sterile inflammation and septic lesions in rabbits, obtained at 1,4, and 24 h post IV administration of 74 MBq of Tc-99m-ceftriaxone. Sterile inflammation at (a) 1 h, (c) 4 h, and (e) 24 h; septic lesions at (b) 1 h, (d) 4 h, and (f) 24 h. For image (c), the oval represents the inflammation site, while in the case of image (d), the left oval represents the infectious lesion and the right oval represents the contralateral side (control).

Table 3

Tissue distribution in normal mice at 1,4, and 24 h after intravenous administration of 100 ml of Tc-99m-ceftriaxone (40 kBq). Data expressed as percent injected dose per whole organ ± SD of three animals

Percent injected dose/whole organ

Figure 3. Blood clearance study of Tc-99m-ceftriaxone in rabbits (average data of three rabbits).

1h 4h 24 h

Blood 14.20 ± 2.1 10.2 ± 0.87 4.9 ± 0.20

Heart 1.22 ± 0.03 0.6 ± 0.04 0.3 ± 0.01

Lungs 2.80 ± 0.04 1.5 ± 0.08 0.4 ± 0.01

Liver 8.6 ± 0.80 14.8 ± 1.16 5.2 ± 0.52

Kidneys 12.2 ± 1.00 15.8 ± 1.22 12.6 ± 1.56

Intestines 2.32 ± 1.36 15.38 ± 2.32 18.2 ± 4.28

Stomach 0.4 ± 0.10 0.8 ± 0.30 0.5 ± 0.21

Table 4

Target to non-target ratio of Tc-99m-ceftriaxone in animal models of sterile inflammation and bacterial infection

Time (h) Tc-99m-CRO

Inflammation Bacterial infection

1 1.6 2.5

2 1.8 3.2

4 1.5 3.6

24 1.4 4.5

CRO, ceftriaxone.

reconstitute the kit will make it very valuable among other kits available in the nuclear medicine field.

The idea here was to label an antibiotic and thereby enable the accurate diagnosis of microorganisms at the site of infection in the routine clinical setting. The approach with Tc-99m-labeled ciprofloxacin has shown considerable promise in preliminary studies, but other studies in animal models and in clinics have shown the ciprofloxacin tracer to disappear from the site of infection as well as from inflammation with equal rapidity.21-23

Recently, we successfully formulated an isoniazid antibiotic derivative as a radiolabeled agent for detecting tubercular infectious lesions.24 In this study CRO was chosen, which is a third-generation cephalosporin antibiotic with a broad spectrum of activity against Gram-positive and Gram-negative bacteria and especially excellent activity with a low minimum inhibitory concentration (MIC) against all the strains of S. aureus.25 This

antibiotic acts by binding to the bacterial wall, inhibiting the synthesis of peptidoglycan (absent in eukaryotes), and inhibiting the synthesis of the bacterial wall leading to bacterial death.26 Moreover, it contains various groups (Figure 1b) to link to a radioisotope such as the extensively used technetium.27

The results obtained in this study show that CRO was successfully labeled with Tc-99m with a high radiochemical yield (Table 1). During labeling of ciprofloxacin, a good amount of R/H Tc-99m is formed, which requires filtration of the preparation before use, as documented in the literature.28 In contrast, Tc-99m-CRO required no such filtration as it contained negligible R/H Tc-99m. The duration and temperature of storage did not show any difference in the labeling yield of the kit, which is a further benefit of this single-vial kit preparation in the nuclear medicine setting in the hospital. The radiotracer exhibited good in vitro and in vivo stability, and a slow release (approximately 6-8%) of Tc-99m-pertechnetate was observed up to 24 h.

The distribution of Tc-99m-CRO showed 90% protein binding, which is in line with reported data.29 The normal Tc-99m-CRO image showed high uptake by the kidneys and in bile, with moderate uptake by the liver and spleen. No uptake by bone, bone marrow, or other soft tissues was observed, and Tc-99m-CRO predominantly showed high blood pool activity. The gall bladder could be seen occasionally, and some bowel activity occurred in the delayed images, which is an important observation in biodistribution studies. The high protein binding explains the higher activity in the blood pool. The radiolabeled CRO followed the same pharmacokinetic profile as the unlabeled antibiotic, without any change in its in vivo behavior. Furthermore, the

Table 5

Clinical data for the ceftriaxone for infection imaging kit used in 36 patientsa

Patient No. Age Sex Tc-99m-MDP scan Tc-99m-CRO scan Microbial culture Remarks

1 21 F + + + True-positive

2 62 M + + + True-positive

3 37 M + + + True-positive

4 48 F + - + (TB) False-negative

5 51 F + + + True-positive

6 60 M + + + True-positive

7 23 M + + + True-positive

8 40 F + + - False-positive

9 39 F + + + True-positive

10 18 M + + + True-positive

11 33 M + + + True-positive

12 27 M + - - True-negative

13 51 F + - - True-negative

14 33 F + + + True-positive

15 42 M + + + True-positive

16 61 M + + + True-positive

17 45 F + - - True-negative

18 78 F + - - True-negative

19 24 M + - + False-negative

20 71 M - - + False-negative

21 70 M + + + True-positive

22 66 M + + + True-positive

23 63 M + + + True-positive

24 59 M + + + True-positive

25 38 M + + + True-positive

26 65 F + + + True-positive

27 49 M + + + True-positive

28 44 M + - - True-negative

29 19 M + + + True-positive

30 70 F + + + True-positive

31 68 M + + + True-positive

32 70 M + + + True-positive

33 52 F + - - True-negative

34 35 F + - - True-negative

35 52 F + + - False-positive

36 24 M + - + False-negative

MDP, methylene diphosphonate; CRO, ceftriaxone; F, female; M, male; TB, tuberculosis. a Sensitivity 85.2%; specificity 77.8%; positive predictive value 92.0%; negative predictive value 63.6%; diagnostic accuracy 83.3%.

Figure 6. Representative multimodality scan showing a non-healing ulcerative lesion in the right leg of an 18-year-old patient with a positive microbial culture report. (a) SPECT image; (b) functional image, coronal slice; (c) computed tomography image, coronal slice showing bone destruction; (d) multimodality fusion of SPECT and computed tomography images documenting the bone and soft tissue infection.

absence of radioactivity in the stomach confirmed the in vivo stability of the radiolabel throughout the duration of the study.

The qualitative scintigraphy evaluation of the radiolabel suggests that the nonspecific uptake of Tc-99m-CRO did not occur at any time point (1,4, and 24 h) in the case of sterile inflammation. In the quantitative ROI analysis of the radiolabeled antibiotic, there was increased uptake of radioactivity and a significantly high target to non-target ratio at the infectious lesion when studied up to 24 h (Table 4). There are several factors that may account for this observation. SAR studies in a series of fluoroquinolones showed that the probable reason for the absence of specificity of ciprofloxacin to staphylococcal infections lies in its structure, which lacks a p-substituted derivative with electron donor properties at the 7-pyyrolidinyl ring for better potency against Staphylococcus.30 However, the presence of the 3-thiotriazine ring in the CRO molecule augments metabolic stability and protein binding, thereby increasing its circulation time, while its 7-aminothiadiazole alkoxy imine group ensures extended beta-lactamase stability and enhanced anti-staphylococcal activity.31 The interaction of Tc-99m-ciprofloxacin with various physiological factors prior to its interaction with the bacterial chromosomes32 and its lesser protein binding (30%) causes reduced availability in the blood pool. Meanwhile, in the case of Tc-99m-CRO, high protein binding results in the increased availability of circulating drug and consequently it continues to accumulate at the site of infection.

The successful imaging of bacterial infection in our study stems from a combination of the direct binding of Tc-99m-CRO to dividing bacteria and the dynamic specificity that occurs when uptake caused by bacterial infection persists and increases with time. Therefore, the results confirm our proposition that Tc-99m-CRO could in fact distinguish aseptic from septic inflammatory processes. We successfully extended its use to the imaging of bacterial infections in human subjects after receiving approval from the human ethics committee.33 Biodistribution studies in human subjects indicated that two-thirds of the radiotracer was excreted through the renal route and a third through the hepatobiliary route, which corroborates the experimental animal studies performed using this preparation and the in vivo behavior

of the antibiotic in the product monograph of the drug itself. Also, when used in some of the patients with clinical and bone scintigraphy evidence of microbial infection, especially in cases of bacteria-induced osteomyelitis,34 the results showed a progressive increase in radiolabeled antibiotic uptake in the involved site, with a high target to non-target ratio at 1 and 4 h post-injection.

The single-vial kit preparation of Tc-99m-CRO has reasonably good accuracy in the clinical diagnosis of orthopedic infections (Figure 6). The use of this formulation is being further validated in a large number of patients and also in a few treated cases of osteomyelitis.

In conclusion, the formulated kit is handy to use and has the potential to improve the early detection and treatment of a wide variety of deep-seated bacterial infections. It also has the ability to localize infective foci accurately, which is important for surgical interventions such as the drainage of abscesses. In addition, serial imaging with Tc-99m-CRO might be useful for monitoring the clinical response to treatment and for optimizing the duration of antimicrobial treatment.

Acknowledgements

We thank the Director, Dr R.P. Tripathi, INMAS (Defence Research and Development Organization) for providing excellent research facilities. This work is supported by Project 1NM-311.

Ethical approval: All animal protocols were approved by the institutional animal ethics committee (8/GO/a/99/CPCSEA).

Conflict of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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