Introduction

Many techniques are available for diagnosis of infection and inflammation. These techniques are X-ray, ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI). But it is well known that these are not the best of techniques for the imaging of infection at early stages [1]. The early detection of the infectious focus by radionuclide imaging helps both patient and medical operators and reduces the time and cost of treatment A number of infection imaging radiotracer were reported including 67Ga-citrate [2, 3], 99mTc or 111In-labelled leukocytes [4], 99mTc-nano-colloid [5], 99mTc or 111In labelled HIG (human polyclonal immunoglobulin) [6, 7] and 99mTc-ubiquicidin 29-4 [8–10]. However, none of the above radiotracer is capable of differentiate in a clinically useful manner between septic and aseptic inflammation [11].

99mTc labelled ciprofloxacin (fluoroquinolone antibiotic) has been proposed for infection imaging because of its simple binding procedure, immovability and fitting scintigrahic results. 99mTc-ciprofloxacin is not the best radiotracer for infection imaging because it has many disadvantages related to radiochemical purity and stability [12–15]. The previous reported data about specificity of 99mTc-ciprofloxacin kit is contradictory [13, 16–22]. Other flouroquinolon derivatives such as sparfloxacin [23], enrofloxacin, levofloxacin [24], pefloxacin [25], lomefloxacin [16], difloxacin [26], moxifloxacin [27], sitafloxacin [28], rifampicin [29] and norfloxacin [30] are labelled with 99mTc to be used for infection imaging and to differentiate between septic and aseptic inflammation. Most of the above 99mTc-fluoroquinolone can not distinguish infection from sterile inflammation. In this study, a new quinolone derivative (7-bromo-1,4-dihydro-4-oxo-quinolin-3-carboxylic acid) was synthesized, well characterized and it is labelling with 99mTc was investigated. Factors affecting the labeling yield of 99mTc-BDOQCA complex and biological distribution in inflammation bearing animals were studied in detail.

Experimental

Melting points were determined on Mettler FP 80 melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded on Perkin Elmer FT-IR Spectrum BX Spectrometer at Cm−1 scale using KBr discs.1H-NMR and 13C-NMR were carried out on Bruker AC 500 MHz and JEOL 300 MHz Spectrometer using TMS as internal stander and chemical shift values were recorded in ppm on δ scale. The 1HNMR data were represented as follow: chemical shifts, multiplicity (s. single, d. doublet, t. triplet, q. quartet, b. broad) and number of protons. 13CNMR data were represented as chemical shifts and type of carbon. EI mass spectra were determined on LC/MS/MS, 3200QTRAP, AB Applied Biosystems. MDS SCIEX, Cairo, Egypt. Thin layer chromatography was performed on precoated (0.75 mm) silica gel GF254 plates (E. Merck, Germany). Visualization was performed by illumination with UV light source (254 nm). BDOQCA was prepared using the modification of previously published procedure [31–35] as described below (Fig. 1).

Fig. 1
figure 1

Reaction scheme for the synthesis of BDOQCA

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Synthesis of 7-bromo-3-carbethoxy-1,4-dihydro-4-oxo-quinoline (3)

Mixture of m-bromoaniline (1.7 g, 0.01 mol) and diethyl ethoxymethylene malonate (2.6 g, 0.01 mol) in absolute ethanol (10 ml) was refluxed for 6 h and left over night. The formed anilide was added to boiling phenyl ether (Ph2O ≈ 200 ml) with stirring and the mixture was refluxed for 90 min and cooled to room temperature. The resulting 3-carbethoxy-4-hydroxy-7-bromoquinoline was filtered, washed several times with a 2:1 mixture of ethyl acetate and petroleum ether and recrystallized from 70% ethanol to afford 2.1 g (71%). mp > 250 IR (KBr): υ (cm-1) 3447.80, 3085.42, 2924, 2853.02, 1773.82, 1700.11, 1654.32, 1193.79; 1H NMR (CDCl3): δ 1.51 (t, 3H, –CH2 CH 3 ), 4.65 (q. 2H, CH 2 CH3), 8.03 (d, J = 10 Hz, 1H, Har), 8.42 (s, 1H, Har), 8.45–8.47 (d, J = 10 Hz, 1H, Har), 9.37 (s, 1H, N–CH = C), 14.5 (b, 1H, –NH); 13C NMR (CDCl3): δ 13.47 (–CH2 CH 3 ), 65.06 (CH 2 CH3), 105.06–146.49 (8 Car), 167.63 (–CO–), 174.25 (–COO–); MS m/z (%): 298 (M+ +2), 252 (100); Anal. (C12H10BrNO3) C, H, N.

Synthesis of 7-bromo-1,4-dihydro-4-oxo-quinolin-3-carboxylic acid (BDOQCA)

A solution of 3-carbethoxy-4-hydroxy-7-bromoquinoline (1 g) in glacial acetic acid (10 ml), water (10 ml) and hydrochloric acid (10 ml) was heated on steam bath for 8 h. After cooling the solution was diluted with water and filtered, the precipitate was recrystalized from ethanol to afford 0.86 g (93%). mp > 250 IR (KBr): υ (cm-1) 3481–2363, 3068,1773.68, 1733.98, 1618, 1222; 1H NMR (CDCl3): δ 8.07–8.09 (d, J = 7.5 Hz, 1H, Har), 8.35 (s, 1H, Har), 8.47–8.49 (d, J = 10 Hz, 1H, Har), 9.3 (s, 1H, –N–CH = C), 13.6 (b, 1H, –OH); 13C NMR (CDCl3): δ 104.06 (C3), 110.9)(C8), 113.2–115.5 (C4`), 117.7–118.8 (C6), 126.2–129.7 (C7), 134.2 9 C5), 140.03 (C8`) 146.8 (C2), 169.7 (–COOH), 174.25 (C4); MS m/z (%):268 (M+, 100), 270 (M+ +2, 98); Anal. (C10H6BrNO3) C, H, N.

Chemicals

All other chemicals were purchased from Merck and they were reactive grade. In all cases, the water used is deoxygenated bidistilled water.

Labeling of BDOQCA by 99mTc

Labeling procedure: BDOQCA 0.5–3 mg (with a 0.5 mg increment in each vial) were taken separately in nitrogen filled vials with 5–35 μg of SnCl2·2H2O with a 5 μg increment in each vial). After gently swirling, 400 MBq of Na99mTcO4 − was added through sterilized syringes to the above preparations. The pH of the preparations was kept from 3.0 to 9.0 (with a 1 increment in each vial) followed by incubation at room temperature and filtration through Millipore filter before investigation.

Analysis

For each labeling experiment, two drops of the reaction product were spotted on two ‘Whatman no. 1’ ascending chromatographic paper strips (each of 10 × 1.5 cm2). One strip was developed with acetone and other strip was developed with ethanol: water: ammonium hydroxide mixture (2:5:1), After complete development, the two radiochromatograms were dried, cut into 0.5 cm pieces and separately counted using the NaI(Tl) scintillation counter to determine the ratio of the hydrolyzed 99mTc, free 99mTcO4 − and 99mTc-complex. It was further confirmed by a Shimadzu HPLC system, which consists of pumps LC-9A, Rheodyne injector and UV spectrophotometer detector (SPD-6A) operated at a wavelength of 320 nm. Chromatographic analysis was performed by injection of 10 μl from the reaction mixture of 99mTc-quinolon into a reversed-phase column (Lichrosorb RP18, 4 mm × 250 mm; 5 μm). The column was eluted with 10% ethanol in 0.2 M phosphate buffer pH 7.2 and the flow rate was adjusted to 0.5 ml/min. Then fractions of 0.5 mL were collected separately using a fraction collector up to 20 mL and counted in a well-type γ-scintillation counter.

Stability of 99mTc-BDOQCA in serum

Stability of 99mTc-BDOQCA was studied in vitro by mixing 1.8 mL of normal serum and 0.2 mL of 99mTc-complex and incubated at 37 °C for 24 h. Exactly 0.2 mL aliquots were withdrawn during the incubation at different time intervals up to 24 h and subjected to ITLC for determination the percent of 99mTc-complex, reduced hydrolyzed technetium and free pertechnetate.

In vitro binding with Escherichia coli

In vitro binding behviour of the 99mTc-BDOQCA complexes was investigated using the reported method [36]. Briefly, 10 MBq of the 99mTc-BDOQCA complex in 0.1 mL of sodium phosphate buffer (Na-PB) was transferred to a clean and sterilized test tube. Thereafter, 0.8 mL of 50% (v/v) 0.01 M acetic acid in a Na-PB containing approximately 1 × 108 colony forming units (CFU) of E. coli were added. The mixture was then incubated at 4 °C for 1 h. The mixture after 1 h was centrifuged for 5 min at 2000 rpm. The bacterial pellets were resuspended after the removal of supernatant in 1 mL Na-PB and recentrifuged. The E. coli pellets after removal of the supernatant analyzed for % activity using well counter.

Induction of infectious foci

A single clinical isolation of E. coli from biological samples was used to produce focal infection. Individual colonies were diluted in order to obtain turbid suspension. Groups of three mice were intramuscularly injected with 200 mL of the suspension in the left lateral thigh muscle [37, 38]. Then, the mice were left for 24 h to get a gross swelling in the infected thigh.

Induction of non-infected inflammation

Sterile inflammation was induced by injecting 200 mL of turpentine oil [39]. Sterilized by autoclaving at 121 °C for 20 min, intramuscularly in the left lateral thigh muscle of the mice. Two days later, swelling appeared.

Induction of heat killed E. coli non-infected inflammation

Sterile inflammation was induced by injecting 200 mL of heat killed E. coli, sterilized by autoclaving at 121 °C for 20 min, intramuscularly in the left lateral thigh muscle of the mice. Two days later, swelling appeared.

The study was approved by the animal ethics committee, Labeled Compound Department, and was in accordance with the guidelines set out by the Egyptian Atomic Energy Authority.

The animals were intravenously injected with 100 μL (100–150 MBq) 99mTc-BDOQCA via the tail vein and kept alive in metabolic cage for different intervals of time under normal conditions. The mice were sacrificed at 2, 4 and 24-hour post-injection. Samples of fresh blood, bone and muscle were collected in pre-weighed vials and counted. The different organs were removed, counted and compared to a standard solution of the labeled BDOQCA. The average percent values of the administrated dose/organ were calculated. Blood, bone and muscles were assumed to be 7, 10 and 40%, respectively, of the total body weight [40]. Corrections were made for background radiation and physical decay during experiment. Both target and non-target thighs were dissected and counted. Differences in the data were evaluated with the Student’s t test. Results for P using the 2-tailed test are reported and all the results are given as mean ± SEM. The level of significance was set at P < 0.05.

Results and discussion

Like other quinolones, BDOQCA has only one possibility to form complex between 99mTc and donating atoms where the chelation was formed between 99mTc and two molecules of BDOQCA each one share with two donor oxygen atoms (one of carbonyl oxygen and other of carboxyl oxygen) [14, 24, 43].

Radiochemical purity and stability of 99mTc-BDOQCA complex were assessed by thin layer chromatographic method and reversed phase high performance liquid chromatography (HPLC). In thin layer chromatography using acetone as the solvent, free 99mTcO4 − moved with the solvent front (Rf = 1), while 99mTc-BDOQCA and reduced hydrolyzed technetium remained at the point of spotting. Reduced hydrolyzed technetium was determined by using ethanol: water: ammonium hydroxide mixture (2:5:1) as the mobile phase, where reduced hydrolyzed technetium remains at the point of spoting (Rf = 0) while other species migrate with the solvent front (Rf = 1). The radiochemical purity was determined by subtracting the sum of the percent of colloid and free pertechnetate from 100%.

An HPLC radiochromatogram was presented in Fig. 2 and showed two peaks, one at fraction No.5, which was corresponds to99mTcO4 −, while the second peak was collected at fraction No.12.9 for 99mTc-BDOQCA which was found to coincide with the UV signal.

Fig. 2
figure 2

HPLC chromatogram of 99mTc-BDOQCA

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Effect of substrate concentration

Figure 3 shows that, the radiochemical yield of 99mTc-BDOQCA complex increased from 38.4 ± 0.76 to 93.7 ± 1.87% by increasing the amounts of BDOQCA from 0.5 to 2.5 mg and the main impurities was colloid (51 ± 2.25% at 0.5 mg BDOQCA). This low labeling yield was due to the substrate concentrations being insufficient to form complex with all of the reduced technetium so, the remained reduced 99mTc were converted to colloid. By increasing the substrate concentration over the optimum values (2.5 mg), the labeling yield was slightly decreased which reach to 93 ± 1.86% at 3 mg BDOQCA.

Fig. 3
figure 3

Variation of the radiochemical yield of 99mTc-BDOQCA as a function of BDOQCA concentration; reaction conditions; x mg BDOQCA, 25 μg of SnCl2·H2O, 0.5 mL (~ 400 MBq) of 99mTcO4 − at pH 6, the reaction mixture was kept at room temperature for 30 min

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Effect of SnCl2·2H2O concentration

As shown in Fig. 4, 25 μg SnCl2·2H2O is the optimum amount at which a maximum labeling of 97.3 ± 1.87% was obtained. Below this value, stannous chloride is not sufficient for complete reduction of pertechnetate to form 99mTc-complex; this is an explanation of the presence of high percentage of free pertechnetate (39 ± 0.31% at 5 μg SnCl2·2H2O). By increasing the amount of SnCl2·2H2O above 25 μg, the radiochemical yield was decreased (88 ± 1.76% at 35 μg SnCl2·2H2O) while the amount of colloid increased and reach to 11 ± 0.07%. This may be due to the fact that most of the ligand molecules were consumed in the formation of complexes, so the pertechnetate is reduced to insoluble technetium (IV) TcO2·xH2O in the absence of ligand [41] or due to the fact that the excess amount of stannous chloride leads to the formation of stannous hydroxide colloid Sn(OH) −3 in basic medium [42].

Fig. 4
figure 4

Variation of the radiochemical yield of 99mTc-BDOQCA as a function of SnCl2·2H2O concentration; reaction conditions; 2.5 mg BDOQCA, X μg of SnCl2.H2O, 0.5 mL (~ 400 MBq) of 99mTcO4 − at pH 6, the reaction mixture was kept at room temperature for 30 min

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Effect of pH of the reaction mixture

As shown in Fig. 5 the percentage yield of 99mTc-BDOQCA increased gradually with increasing the pH up to 6 to give a labeling yield of 97.3 ± 1.87% at 30 min. At lower pH values, the yield was decreased till reached to 65.4 ± 1.3% at pH 3 where free pertechnetate was the main impurities. At pH above the optimum value, the radiochemical yield is drastically decreased (45.5 ± 0.9% at pH 9) by forming RH-99mTc which is the main radiochemical impurities (48.5 ± 0.34% pH 9) where alkaline medium is a good medium for colloid formation.

Fig. 5
figure 5

Effect of pH of the reaction medium on the radiochemical yield of 99mTc-BDOQCA complex; reaction conditions; 2.5 mg BDOQCA, 25 μg of SnCl2.H2O, 0.5 mL (~ 400 MBq) of 99mTcO4 − at pH = X, the reaction mixture was kept at room temperature for 30 min

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Effect of reaction time and stability test:

The stability of 99mTc-complex was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of the labeled compound. These undesired radioactive products may be accumulated in non-target organs. Figure 6 shows the rate of formation of 99mTc-BDOQCA complex started relatively slowly with a yield of 80.1 ± 1.6% at 2 min reaction time. The yield was increased with time till the maximum yield of 97.3 ± 1.87% which achieved at 30 min reaction time. The formed complex was stable for a time up to 8 h.

Fig. 6
figure 6

Effect of the reaction time on the radiochemical yield of 99mTc-BDOQCA; reaction conditions; 2.5 mg BDOQCA, 25 μg of SnCl2.H2O, 0.5 mL (~ 400 MBq) of 99mTcO4 − at pH 6, the reaction mixture was kept at room temperature and at different reaction time

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Stability in serum

As shown in Fig. 7, incubation of the preparation containing 99mTc-BDOQCA in normal serum for 24 h at 37 °C resulted in a small release of radioactivity (8.6 ± 0.6) from the 99mTc-BDOQCA, as determined by HPLC and ITLC.

Fig. 7
figure 7

Stability of 99mTc-BDOQCA in normal serum as a function of time

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In vitro binding with E. coli

Competition binding of the 99mTc-BDOQCA to E. coli was assessed by pre-incubating the bacteria with 10–100 fold excess of the unlabeled corresponding BDOQCA and then assessing the amount of radioactivity bound to the bacteria. Adding additional cold BDOQCA significantly decreases the binding of 99mTc-BDOQCA to living bacteria indicating that; 99mTc-BDOQCA complex is a specific agent for bacterial cells. The in vitro binding affinity of the 99mTc-BDOQCA complexes is shown in Fig. 8.

Fig. 8
figure 8

In vitro binding of the 99mTc-BDOQCA to E. coli

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Biodistribution

The uptake of the 99mTc-BDOQCA complex in different organs of the animals infected with living, heat killed E. coli and turpentine oil is given in Table 1. The uptake of 99mTc-BDOQCA was significantly low in heat killed E. coli and turpentine oil infected group of animals (aseptic inflammation) as compared to infected group with living bacteria (abscess). These data depicted rapid distribution throughout the body and uptake in the inflamed areas was observed within 2 h after intravenous injection of the tracer. As shown in Fig. 9, mice with infectious lesions injected with 99mTc-BDOQCA showed a mean abscess-to-muscle (target-to-non target, T/NT) ratio equal to 7.6 ± 0.6, after 2 h post injection. 99mTc-BDOQCA shows higher T/NT in the infected muscle (live E coli) at all time intervals than that of sterile inflamed muscle (heat killed E. coli and turpentine). This 99mTc-BDOQCA showed higher uptake in infected tissue than 99mTc-ciprofloxacin (T/NT = 3.6 ± 0.4). The mean abscess-to-muscle (T/NT) ratio for 99mTc-BDOQCA was higher than that of other recently published 99mTc-labeled antibiotics such as sparafloxacin (T/NT = 5.9 ± 0.7) [23], difloxacin (T/NT = 5.5 ± 0.5) [26], norfloxacin (T/NT = 6.9 ± 0.4) [30], rifampicin (T/NT = 7.3 ± 0.7) [29], ceftraixone (T/NT = 5.6 ± 0.6) [43], streptomycin (T/NT = 2.4 ± 0.1) [44], and N-sulfanilamide (T/NT = 2.9 ± 0.1) [45].

Table 1 Biodistribution of 99mTc-BDOQCA in E. coli, heat killed E. coli and turpentine oil inflamed mice at different time intervals
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Fig. 9
figure 9

The ratio of target muscle (T) to non-target muscle (NT) of 99mTc-quinoline (left) in different inflammation models at different post injection times

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Conclusion

In this study, BDOQCA was synthesized, well characterized and labeled with 99mTc with a labeling yield of 97.3% using simple and instantaneous method. 99mTc-BDOQCA shows higher mean abscess-to-muscle (target-to-non target, T/NT) ratio in the infected muscle (live E. coli) at all time intervals than that of sterile inflamed muscle (heat killed E. coli and turpentine). 99mTc-BDOQCA complex showed higher labelling yield, stability and uptake in infected tissue (T/NT = 7.6 ± 0.6) than the commercially available 99mTc-ciprofloxacin (T/NT = 3.6 ± 0.4) [21]. These results were promising enough to state that 99mTc-BDOQCA could be used instead of the commercially available 99mTc-ciprofloxacin as a good radiotracer for imaging of infection at early stages and distinguishing infection from sterile inflammation.