Synthesis and evaluation of Ciprofloxacin derivatives ...

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Synthesis and evaluation of Ciprofloxacin derivatives as diagnostic tools for bacterial infection by Staphylococcus aureusw Saurabh Dahiya,abc Krishna Chuttani,b Roop K. Khar,c Daman Saluja,a Anil K. Mishra*b and Madhu Chopra*a Received 28th April 2009, Accepted 30th June 2009 First published as an Advance Article on the web 5th August 2009 DOI: 10.1039/b908474f Development of target-specific diagnostic radiopharmaceuticals has always been a challenging task. For this purpose, design and development of the imaging-friendly variant of a potent antibiotic could aid in treatment planning and follow-up of patients with hard-to-diagnose bacterial infections. Fluoroquinolone analogues were synthesized taking the lead from Ciprofloxacin (the broad spectrum antibiotic) molecule. The idea of modifying fluoroquinolones, and subsequently labeling them, was to preserve their capacity to bind bacteria and thereby enable the compound to specifically target those microorganisms. Three compounds were thus synthesized as derivatives of Ciprofloxacin. The fluoroquinolone analogues were labeled with 99m Tc by using 99mTc pertechnetate with high labeling efficiency for all the formulations. The complexes formed by chelation of 99mTc with our synthesized fluoroquinolone analogues showed good in vitro serum stability. The blood clearance study performed in New Zealand White rabbits exhibited a curve indicating the initial fast phase in which radiocomplexed drugs cleared from blood very quickly followed by a slow phase. The in vivo evaluation showed that fluoroquinolone-based radiopharmaceuticals bind to the bacteria present at the site of infection, which results in the retention of the agent at sites of active bacterial infection. The biodistribution data and the scintigrams demonstrated that Staphylococcus aureus bacteria in animal infection models took up the radiopharmaceutical formulations, confirming our hypothesis that 99mTc fluoroquinolone derivatives might be useful as diagnostic agents for targeted delivery in bacterial infections.

A.

Introduction

In spite of the great strides in the management of infectious diseases, infections remain among the most frequently encountered and costly causes of deaths and diseases worldwide, particularly in developing countries.1 Timely diagnosis of infectious ailments could help in instituting effective treatment and reduce morbidity and mortality.1,2 Accurate diagnosis to enable appropriate treatment is therefore central to strategies employed in the ongoing struggle against microbial infection.3 Using radiopharmacy techniques and nuclear medicine, drug molecules and carrier systems may be radiolabeled and their release, biodistribution and uptake may be visualized in human subjects/animal models. The property of selective toxicity (i.e. destroying the microbe but causing little harm to the patient) is the basis of the use of antimicrobial compounds, e.g. antibiotics, to treat infections and has been a

Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007, India. E-mail: [email protected], [email protected]; Fax: +91-11-27666248; Tel: +91-11-27666272 b IDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. SK Mazumdar Road, Delhi-54, India. E-mail: [email protected] c Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India w Electronic supplementary information (ESI) available: HPLC, NMR and MS spectra of compounds 2–9. See DOI: 10.1039/b908474f

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The Royal Society of Chemistry 2009

life saving in this respect. The same property provides the potential for these agents to be exploited as radiopharmaceuticals for infection imaging. The principle is simple: the specificity for infection is provided by the antimicrobial agent binding to the microbe, which in turn can be visualized by a gamma camera because the antimicrobial agent is labeled with a gamma emitter such as 99mTc.3 The theoretical advantage of using an antimicrobial agent as the localizing agent for infective foci is the selective toxicity of the compound for microbial rather than human targets. The labeling of antibiotics was introduced in 1993 by Vinjamuri et al. in their search for a better agent to diagnose infection.4 Ciprofloxacin, marketed in 1987, was the first widely administered quinolone with advanced systemic activity. Second-generation agents, now called fluoroquinolones, have excellent activity against many Gram-negative aerobes. With the advent of this drug class, clinicians were able for the first time to treat a wide range of Gram-negative aerobic infections orally; fluoroquinolones thus constituted a significant advancement in the management of infectious diseases.5 The bactericidal activity generated by fluoroquinolones is caused by their inhibition of bacterial DNA gyrase and topoisomerase IV enzymes. DNA gyrase is essential for the replication, transcription, and repair of bacterial DNA, and topoisomerase IV is involved in the partitioning of chromosomal DNA during cell wall division. By inhibiting those enzymes, fluoroquinolones keep cellular bacterial DNA in a Metallomics, 2009, 1, 409–417 | 409

supercoil state, thereby preventing bacterial replication.6 Ciprofloxacin also binds to the equivalent mammalian enzyme, topoisomerase II, but with 100 to 1000 times lesser affinity and the binding is readily reversible.3 Ciprofloxacin is retained at sites of infection, giving high target to background ratio and permitting infection-specific imaging to occur when sequential images are taken at 1, 4 and, if required, 24 hours post injection. It associates freely with metal ions, allowing it to be radiolabeled with technetium. Infection imaging is perhaps the most prolific area for radiopharmaceutical development and a survey of the literature would surely result in dozens of potentially ‘useful’ new products, but few, if any, have stood the test of time. The main challenges in infection imaging are the ability to distinguish true infection from sterile inflammatory processes and the need for a universal detector of inflammation/infection to replace the use of radiolabeled white cells.7 An ideal infection-imaging agent should have following features: (1) efficient accumulation and good retention in infectious foci, (2) rapid clearance from the background, (3) no accumulation in non-target organs, (4) no toxicity, no immune response, (5) early diagnostic imaging, (6) ready availability and low cost, (7) low radiation burden, (8) low-hazard preparation, (9) differentiation between infection and non-microbial inflammation.8 The radiopharmaceuticals developed in the present study were designed to provide answers to some problems such as specificity, low affinity and higher doses of 99mTc-Ciprofloxacin required for infection imaging. The new compounds were developed by keeping the basic skeleton of Ciprofloxacin intact and substituting it with a metal chelator to possibly find a new molecule with high affinity for bacterial infection. The compounds were developed by modifying two available reactive groups present on the molecule and the effect of substitution was studied in its targeting capacity in in vivo models.

B. 1

Experimental Materials & methods

Ciprofloxacin hydrochloride USP, as a gift sample for research work, was received from SUN Pharmaceuticals Ltd, Silvassa (UT of D & NH), India. Chemicals and reagents were of analytical grade and were procured from both international and local suppliers like Sigma Aldrich, Fluka, Merck, S. D. fine, Qualigens, Thomas Baker and CDH. The 1H NMR spectra were obtained by using Bruker-Avance II-400 at INMAS & Jamia Hamdard and tetramethylsilane was used as internal standard. IR spectra were obtained by using Shimadzu instrument model 8300 and mass spectra were recorded on a Q STAR XL MS system, Applied Biosystems at ACBR, University of Delhi and Agilent 6310 Ion Trap at INMAS, Delhi. The radioactivity counter was a gamma ray counter (Type GRS23C, Electronics Corporation of India Limited, Mumbai, India). Animal handling and experimentation was carried out as per the guidelines of the Institutional Animal Ethics Committee. Stocks of the bacterial strain Staphylococcus aureus were obtained from the Vallabhbhai Patel Chest Institute (University of Delhi). 410 | Metallomics, 2009, 1, 409–417

2 Synthesis & characterization of Ciprofloxacin analogues 2.1 Preparation of compound 2 (Boc-protected Ciprofloxacin). Boc-protection of the NH group of Ciprofloxacin molecule 1, was done by following a general literature14 method. To a solution of Ciprofloxacin hydrochloride (1-cyclopropyl-6fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride 1, 2 mmol, 0.662 g) in a 1 : 1 mixture of THF–H2O (20 mL), NaHCO3 (8 mmol, 0.672 g) and Boc2O (2.4 mmol, 0.523 g) were added consecutively at 0 1C with magnetic stirring. After 30 min, the reaction mixture was allowed to attain room temperature & stirring continued overnight. The turbid solution was extracted with Et2O (2 100 mL). The aqueous layer was acidified to pH = 4 to 5 by careful addition of half saturated cold citric acid and then extracted with CH2Cl2 (3 100 mL). The combined organic phase was dried over anhydrous Na2SO4 and evaporated under reduced pressure to give the Boc-protected Ciprofloxacin, 2 with high purity as a white solid. This was used for the next step without purification. Yield: 0.770 g (90%). IR, nmax (KBr)/cm 1: 3100–2700 (OH), 1733.96 (CO), 1695.67 (CO), 1628.80 (CO), 1248 (CO); NMR: dH (400 MHz, CDCl3, Me4Si) 8.694 (s, 1H), 7.954–7.921 (d, 1H), 7.324–7.306 (d, 1H), 3.606–3.576 (t, 4H), 3.510 (m, 1H), 3.231–3.219 (t, 4H), 1.168–1.150 (m, 1H), 1.430 (s, 9H), 1.295–1.270 (m, 1H). MS (m/e): found 431 (M+), 454 (M+ + Na+), 432 (MH+), 375, 301, 379, 214, (calculated for C22H26FN3O5 = 431.45). 2.2 Preparation of compound 3, (NHS ester of Bocprotected Ciprofloxacin). Boc protected Ciprofloxacin (2, 1 mmol, 0.432 g), NHS (1.1 mmol, 0.126 g) and DCC (1.1 mol, 0.226 g) were mixed in a round-bottomed flask containing 10 mL of dry dichloromethane (DCM) and the reaction mixture was stirred at 4 1C for 48 hours. Dicyclohexylurea (DCU) formed as a by-product was removed by vacuum filtration. The filtrate was dried over anhydrous sodium sulfate and the dichloromethane layer evaporated off yielding 3, the NHS ester of Boc-protected Ciprofloxacin, as a light brown solid. Yield: 0.320 g (60%). IR: nmax (KBr)/cm 1 1790.20, 1734.31, 1696.10, 1627.81 (COs), 1247.97 (C–O). NMR: dH (400 MHz, DMSO, Me4Si) 8.673 (s, 1H, C2–H), 7.954–7.604 (d, 1H, C5–H), 7.585 (s, 1H, C8–H), 3.607–3.586 (t, 4H, 2 CH2), 3.549 (m, 1H), 3.211–3.209 (t, 4H, 2 CH2), 2.588 (s, 4H, 2 CH2), 1.431 (s, 9H, 3 CH3), 1.293–1.256 (m, 4H, 2 CH2). MS (m/e): found 528.80 (M+) (calculated for C26H29FN4O7 = 528.52). 2.3 Preparation of compound 4 (Boc-protected Ciprofloxacin– TETA). To a solution of NHS ester of Boc-protected Ciprofloxacin (3, 0.28 mmol, 0.132 g) in dry DMF (10 ml) was added tetraethylenetetraamine (TETA, 2.8 mmol, 0.409 g) and few drops of dry pyridine in a round-bottomed flask fitted in an oil bath maintained at 80 1C. The solution was stirred for 48 hours. DMF was removed from the reaction mixture upon completion of the reaction, monitored through TLC, at reduced pressure on a Rotavapor. The contents of roundbottomed flask were dissolved in water and extracted with chloroform. Chloroform was evaporated from the organic layer to obtain 4, Boc-protected Ciprofloxacin–TETA. This journal is

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Yield: 0.106 g (68%). IR: nmax (KBr)/cm 1 3421, 3335.79 (Br, NH), 1654.34 (CO), 1627 (NH), 1246.11 (CO). NMR: dH (400 MHz, CDCl3, Me4Si) 9.98–9.96 (Br s, NH), 8.829–8.80 (s, 1H, C2–H), 8.0952–8.0297 (d, 1H, C5–H), 7.3839 (s, 1H, C8–H), 4.037–4.017 (m, 2 CH2), 3.675–3.492 (t, 2 CH2), 3.492 (m, 1H), 3.287–3.1049 (m, 3 CH2), 3.104–3.053 (t, 3 CH2), 1.964–1.924 (m, NH’s), 1.5009 (s, 9H), 1.413 (m, 2H), 1.2909 (m, 2H). MS (m/e): found 558.92 (M+ 1), M+ 31 (calculated for C28H42FN7O4 = 559.67). 2.4 Preparation of compound 5 (Ciprofloxacin–TETA). The Boc-protected Ciprofloxacin–TETA (4, 1 mmol, 0.559 g) was stirred with trifluoroacetic acid (TFA, 1 ml) in a roundbottomed flask at 0 1C in an ice bath for 1 hour. The TFA was evaporated, and ice-cold water and sodium bicarbonate (0.1 g) added to the contents of the round-bottomed flask. The product was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate and the solvent (chloroform) evaporated to obtain compound 5, Ciprofloxacin– TETA as a dry light brownish solid (Scheme 1). Yield: 0.403 g (88%). IR, nmax (KBr)/cm 1 3326.47 (Br, NH), 1654.18 (CO), 1626.54 (CO NMR: dH (400 MHz, CDCl3, Me4Si) 8.817 (s, 1H, C2–H), 8.084–8.014 (s, 1H, C5–H), 7.421–7.39 (s, 1H, C8–H), 4.073–4.012 (m, 2 CH2), 3.685–3.482 (t, 2 CH2), 3.498 (m, 1H), 3.371–3.112 (m, 3 CH2), 3.108–3.063 (t, 3 CH2), 1.960–1.921, (m, NH’s), 1.410–1.300 (m, 2H), 1.258–1.082 (m, 2H). MS (m/e): found 457.4 (M+ 2) (calculated for C23H34FN7O2 = 459.56). Anal. calc. C, 60.11; H, 7.46; N, 21.33; found: C, 60.63; H, 7.85; N, 21.92%. 2.5 Preparation of compound 6 (DTPA-linker). To a threenecked round-bottomed flask, 1,3-diaminopropane (2.8 mmol, 0.207 g) was added and the reaction mixture maintained at 0 1C in an ice bath. Diethylenetriaminepentaacetic (DTPA) anhydride (2.8 mmol, 1.0 g) mixed with dry DMF (15 ml) was added dropwise through a dropping funnel to the reaction mixture. After 4 h, the reaction mixture was brought to room temperature and further heated up to 80 1C in an oil bath and the reaction continued up to 48 h. DMF was evaporated on a Rotavapor at high vacuum to obtain 6, DTPA-linker as a

transparent, crystalline dry product. Yield: 0.930 g (71%). IR: nmax (KBr)/cm 1 3300–2500 (Br, OH & NH), 1716.12 (CO), 1654.12 (NH bend). NMR: dH (400 MHz, D2O) 3.9493–3.8209 (s, 6H, 3 CH2), 3.8209–3.5925 (m, 4H, 2 CH2), 3.4808–3.2772 (m, 8H, 4 CH2), 3.064–3.043 (m, 4H, 2 CH2), 1.99–1.71 (t, 2H). MS (m/e): found 466.35 (MH+ ), 461.02 (M+ 4), (calculated for C17H31N5O10 = 465.45). 2.6 Preparation of compound 7 (Boc-protected Ciprofloxacin– DTPA-linker). To a solution of Boc-protected Ciprofloxacin NHS ester, (3, 1 mmol, 0.530 g) in dry DMF (5 ml), DTPA linker (6, 1 mmol, 0.436 g) and two drops of pyridine were added in a round-bottomed flask maintained at 80 1C in an oil bath. The solution was stirred for 48 hours and the residual solvent (DMF) removed with a Rotavapor by vacuum suction and the precipitated DCU filtered off. The dichloromethane layer of the filtrate was evaporated to obtain 7, Boc-protected Ciprofloxacin–DTPA-linker as off-white solid. Yield: 0.6250 g (62%). IR: nmax (KBr)/cm 1: 3326.74 (NH), 3400–2700 (OH), 1654.03 (CO), 1626.78 (NH bend). NMR: dH (400 MHz, DMSO, Me4Si) 15.197 (Br, OH), 8.674 (s, 1H, C2 –H), 7.954–7.921 (d, 1H, C5 –H), 7.587 (s, 1H, C8–H), 5.583–5.566 (Br, NHs), 3.816 (m, 1H, C –H), 3.401 (s, 10H, 5 CH2), 3.387–3.307 (m, 8H, 4 CH2), 3.259–3.221 (m, 4H, 2 CH2), 2.892–2.736 (m, 8H, 4 CH2), 1.873–1.773 (m, 2H, CH2), 1.433 (s, 9H, 3 CH3), 1.425–1.212 (m, 4H, 2 CH2). MS (m/e): found 857.6 (M+ + Na+ 28) (calculated for C39H55FN8O13 = 862.89). 2.7 Preparation of compound 8 (Ciprofloxacin–DTPA-linker). To a solution of Boc-protected Ciprofloxacin–DTPA-linker (7, 1 mmol, 0.870 g) in a round-bottomed flask maintained at 0 1C was added TFA (1 ml) and the reaction mixture was stirred for 1 hour. TFA was removed from the reaction mixture with a Rotavapor and the contents of the roundbottomed flask neutralized by adding ice cold water and NaHCO3. The contents of the flask were extracted with chloroform and the organic layer evaporated with a Rotavapor to obtain 8, Ciprofloxacin–DTPA-linker as off-white solid (Scheme 2). Yield: 0.650 g (85.5%). IR: nmax (KBr)/cm 1:

Scheme 1 Synthesis of Ciprofloxacin–TETA (compound 5). Reagents: step 1: (BOC)2O, H2O–THF (1 : 1); step 2: NHS, DCC, dry DCM; step 3: TETA, dry DMF; step 4: TFA, 0 1C.

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Scheme 3 Scheme 2 Synthesis of Ciprofloxacin–DTPA-linker (compound 8). Reagents: step 1: DMF; step 2: compound 3, dry DMF, Py; step 3: TFA.

3327.79 (NH), 3450–2750 (OH), 1626.48 (CO), 1576.85. NMR: dH (400 MHz, D2O) 8.341 (s, 1H, C2 –H), 7.781–7.748 (d, 1H, C5 –H), 7.494–7.476 (s, 1H, C8 –H), 3.496 (m, 1H, CH), 3.401 (s, 10H, 5 CH2), 3.387–3.307 (m, 8H, 4 CH2), 3.259–3.221 (m, 4H, 2 CH2), 2.892–2.736 (m, 8H, 4 CH2), 1.873–1.773 (m, 2H, CH2), 1.425–1.212 (m, 4H, 2 CH2). MS (m/z): found 786.57 (MH+) (calculated for C34H47FN8O11 = 762.78). Anal. calc. C, 53.54; H, 6.21; N, 14.69; found: C, 53.43; H, 6.28; N, 14.91%. 2.8 Preparation of compound 9 (Ciprofloxacin–DTPA). To a solution of Ciprofloxacin (1, 1 mmol, 0.331 g) in dry DMF (10 ml) in a three-necked round-bottomed flask was added a mixture of DTPA anhydride (1.2 mmol, 0.428 g) in dry DMF dropwise through a dropping funnel. The reaction mixture was stirred for 24 hours. DMF was evaporated with a Rotavapor under high vacuum to obtain 9, Ciprofloxacin– DTPA as light yellow solid (Scheme 3). Yield: 0.6330 g (92%). IR: nmax (KBr)/cm 1: 3400–2800 (Br, OH), 1730.87, 1628.29 (CO), 599.72 (OH bend). NMR: dH (400 MHz, D2O) 8.329 (s, 1H, C2–H), 7.541–7.507 (d, 1H, C5–H), 6.978–6.973 (d, 1H, C8–H), 4.112 (s, 1H), 3.655 (s, 8H, 4 CH2), 3.587–3.553, (s, 2H, 1 CH2), 3.458–3.398, (m, 4H, 2 CH2), 3.247–3.217, (m, 4H, 2 CH2), 3.011–2.898, (t, 4H, 2 CH2), 2.905–2.874 (t, 4H, 2 CH2), 1.182–1.166 (m, 2H), 0.945–0.852 (m, 2H). MS (m/e): found 701.53 (M+ 5), 702.53 (M+ 4), (calculated for C31H39FN6O12 = 706.64). Anal. calc. C, 52.02; H, 5.38; N, 12.13; found: C, 52.32; H, 5.43; N, 12.34%. 412 | Metallomics, 2009, 1, 409–417

Synthesis of Ciprofloxacin–DTPA (compound 9).

3 HPLC The solvent system was comprised of acetonitrile (solvent A) and premixed filtered and degassed buffer (10 mmol KH2PO4 and 5 mmol (butyl)4NHSO4) in water (pH 3.0, Solvent B). The analysis was performed on reversed Phase C-18 Analytical column (C-18 RP Beckman column 5 mm, 4.6 mm 25 cm). The gradient was set starting with ACN (Solvent A, 100%) to ACN–buffer: 50–50 in 30 minutes. The flow rate was 0.5 mL min 1 and the run was performed for 30 min with a detector set at 278 nm. 4 Radiolabeling with

99m

Tc

The radiolabeling of Ciprofloxacin and its synthesized derivatives was done by dissolving the required amount of drug in 1 ml water for injection in a sterile glass vial. Then a reducing agent such as stannous chloride (Sigma chemicals Co. USA) or stannous tartrate was added and pH was adjusted to neutral or near neutral with appropriate base or acid. The mixture was passed through a 0.22 mm membrane filter (Millipore) into a sterile vial. 1–2 ml of 99mTc as pertechnetate (99mTcO4 ) [extracted from a 99Mo-99mTc generator, by solvent extraction method, supplied by the Board of Radiation & Isotope Technology (BRIT), Government of India] containing 2.0 to 3.0 mCi was added dropwise and incubated at room temperature for 15 to 25 min to achieve optimum radiolabeling efficiency. 5 Radiolabeling efficiency The radiolabeling efficiency of our radiolabeled formulations was ascertained by using Instant Thin Layer ChromatographySilica Gel (ITLC-SG) impregnated strips as stationary phase This journal is

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and acetone as a mobile phase in different experiments. Acetone was found to be the most suitable mobile phase to resolve the species discretely. A direct radiolabeling method was used, and the protocols were standardized to radiolabel the drugs. The optimum concentration of the reducing agents needed for the reaction to achieve optimum radiolabeling efficiency was worked out. Various conditions like the amount of reducing agent, effect of pH, incubation time, and amount of pertechnetate used were standardized to achieve the optimum radiolabeling efficiency.

5.1 Quality control of radiolabeled formulations. The quality of the radiolabeling procedure was checked by determining the percentage of colloids present in the radiolabeled formulations. It was done by the ITLC method using PAW (pyridine, acetic acid, water in the volume ratio 3 : 5 : 1.5 respectively) as the developing solvent. Two narrow strips of ITLC-SG (1 cm 10 cm) were taken. A 2 ml aliquot of sample was applied 1 cm above the lower end of the strip and it was left for a few minutes for the spot to dry. The strips were put in the chromatography chamber of PAW. When the solvent had run two thirds of the strip length, the strip was taken out and air-dried. The strips were cut just above the point of sample application. Both portions of the strips were counted for the radioactivity present therein. 6

In-vitro stability in serum and saline

The in vitro stability of the complexes was checked in normal human serum as well as in normal saline solution. The in vitro serum stability of the complex was estimated in the human serum by incubating 50 ml of the complex-with 450 ml of human serum at 37 1C. Aliquots at different time periods were applied on an ITLC-SG strip and allowed to run in 100% acetone to check any dissociation/degradation of the labeled complex. The dissociation was estimated as the percentage of radiolabeled complex remaining after incubation time intervals of 0 h, 1 h, 2 h, 4 h, 6 h and 24 h. The stability of the radiolabeled complexes in normal saline solution was checked in 100% acetone or 0.9% sodium chloride as developing solvents. This procedure was repeated at different time intervals of 0 h, 1 h, 2 h, 4 h and 24 h for the determination of 99mTc-fluoroquinolone complex stability in saline. 7 Pharmacokinetics of radiolabeled analogues

99m

Tc Ciprofloxacin and its

The blood clearance study was performed in rabbits weighing about 3 Kg, after administering 300 ml of labeled product intravenously through the dorsal ear vein. At time intervals of 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h, about 0.5 ml blood was withdrawn from the dorsal vein of the other ear of the animal. The samples were weighed accurately to the second decimal place in grams and radioactivity was measured using a gamma counter that was calibrated earlier for 99mTc energy. The data were expressed as percent administered dose at each time interval assuming the whole body blood volume as 7% of the body weight. This journal is

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8 Experimental animal infection model Staphylococcus aureus bacteria were grown and maintained as plate cultures and stored at 4 1C. The day before each study, liquid cultures in Luria broth (LB) medium were seeded and grown overnight at 37 1C while shaking at 250 rpm. Bacteria were counted using a haemocytometer and diluted in LB medium for use. To prepare the infection model, aliquots of the bacterial cultures were diluted in LB medium to a concentration of 4.8 108 cells mL 1, and 0.1 mL was administered subcutaneously in one thigh of each of the mice. 9 Animal biodistribution studies Biodistribution studies of labeled fluoroquinolone analogues were studied in 2–3 month old Balb/C mice (normal mice) and infected mice. The mice weighing about 25–30 g were administered intravenously through the tail vein with 3.7 MBq of the radiotracer. The mice were sacrificed at 1, 4 and 24 h post administration and various organs were removed, cleared of adhering tissue, washed with physiological saline, dried in filter paper folds and weighed and the radioactivity in each organ measured using a well-type gamma spectrometer counter. The injected dose was corrected for the retention of administered amount in the tail. The actual amount of radioactivity administered to each animal was calculated by subtracting the activity left in the tail from the activity injected. Radioactivity accumulated in each organ was expressed as percent administered dose per gram of tissue. An amount equal to 7% of body weight was considered to represent the whole-body blood and the data were expressed as the percent administered dose at each time interval. 10 Scintigraphic imaging of the animals (radiotracer localization) The infected mice were injected with 99mTc-Ciprofloxacin, 99m Tc-Ciprofloxacin–TETA (99mTc-5), 99mTc-Ciprofloxacin– DTPA-linker (99mTc-8) and 99mTc-Ciprofloxacin–DTPA (99mTc-9) (0.1 ml; 10 MBq). Planar static images of these rodents were acquired in posterior position for three minutes on the collimator of a gamma camera with their legs stretched out. The procedure was repeated in separate experiments with equivalent doses of all the four fluoroquinolones. Whole body images were taken at 1 h and 3 h post injection of 99m Tc-fluoroquinolone in S. Aureus-infected mice.

C. Results 1 Synthesis of fluoroquinolone compounds Various fluoroquinolone analogues were synthesized taking their lead from the Ciprofloxacin molecule, keeping in mind the chelation process where binding to 99mTechnetium is a prerequisite condition for further radiolabeling, related studies and uses. The compounds thus synthesized according to Schemes 1, 2 & 3 were named as: Ciprofloxacin–TETA (compound 5), Ciprofloxacin–DTPA (compound 8) and Ciprofloxacin–DTPA-linker (compound 9). All the synthesized compounds were characterized by spectroscopic techniques such as IR, NMR, MS and CHN analysis. Metallomics, 2009, 1, 409–417 | 413

2

HPLC

The synthesized compounds were checked for analytical purity using HPLC. The observed retention time for standard Ciprofloxacin was 10.2 minutes, Ciprofloxacin–TETA (5) 11.8 minutes, Ciprofloxacin–DTPA-linker (8) 12.8 minutes and Ciprofloxacin–DTPA (9) 15.7 minutes. The purity of all the compounds was between 90 to 100%. 3

Radiolabeling

Radiolabeling efficiency of formulations of Ciprofloxacin along with three of the synthesized analogues was ascertained by ITLC-SG/SA using acetone as a mobile phase in different experiments. High radiolabeling efficiency for all the formulations was obtained viz.: 99mTc-Ciprofloxacin – 99.54% 0.27%, 99m Tc-Ciprofloxacin–TETA (99mTc-5) – 95.86% 0.50%, 99m Tc-Ciprofloxacin–DTPA (99mTc-9) – 98.48% 0.54% and 99mTc-Ciprofloxacin–DTPA-linker (99mTc-8) – 97.98% 0.42%. 4

Radiochemical purity

Determining the percentage of colloids present in the radiolabeled formulations checked the quality of the radiolabeling procedure. Quality control done with ITLC showed that 99m Tc-Ciprofloxacin, 99mTc-Ciprofloxacin–DTPA (9) and 99m Tc-Ciprofloxacin–DTPA-linker (8) had very low percentages of colloids i.e. less than 1%, while the percentage of colloids present was high i.e. 3.8 0.26% in the case of 99m Tc-Ciprofloxacin–TETA (5). 5

Stability tests in serum and saline solution

The in vitro serum stability of all the four formulations was checked and these were found to be stable up to 24 h of incubation at 37 1C. Up to 9.5% free radiolabel was found at 24 h of incubation in the case of 99mTc-Ciprofloxacin as determined by ITLC, while in formulations 99m Tc-Ciprofloxacin–TETA (99mTc-5), 99mTc-Ciprofloxacin– 99m and Tc-Ciprofloxacin–DTPADTPA (99mTc-9) 99m linker ( Tc-8) it was 11.68% 10.28% and 10.09%, respectively. The in vitro stability of the radiolabeled formulations in normal saline at 37 1C was checked up to 24 h at various time points. For 99mTc-Ciprofloxacin the instant radiolabeling was 96.2%, which increased to more than 99% after an incubation time of 1 h and was more than 95% up to 4 h and remained at 90% at 24 h. In the case of 99mTc-Ciprofloxacin–DTPA (99mTc-9) the instant radiolabeling was 94.9%, which increased to more than 99.2% after an incubation time of 1 h and was more than 92% up to 4 h and 88.5% up to 24 h. For 99mTc-Ciprofloxacin–DTPA-linker (99mTc-8) the instant radiolabeling was 96.2%, which increased to 98.4% after an incubation time of 1 h and was 95.3% up to 4 h and 90.6% up to 24 h. In the case of 99mTc-Ciprofloxacin–TETA (99mTc-5), the instant radiolabeling observed was 92.8%, which increased to 96.5% after an incubation time of 1 h and was 93.89% up to 4 h and declined to 88.6% at 24 h. The radiolabeled complexes were thus found to be stable in vitro up to 24 h in normal saline at 37 1C. 414 | Metallomics, 2009, 1, 409–417

6 Pharmacokinetics The blood clearance study was performed in rabbits weighing about 3 Kg after administering 300 ml of labeled product intravenously through the dorsal ear vein. At different time intervals of 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 24 h; about 0.5 ml blood was withdrawn from the dorsal vein of the other ear of the animal. The samples were weighed accurately to the second decimal place in grams and radioactivity was measured using a gamma counter that was calibrated earlier for 99mTc energy. The data were expressed as percent administered dose at each time interval assuming the whole body blood volume as 7% of the body weight. Blood clearance of the radiolabeled drugs in New Zealand White rabbits exhibited an initial fast phase in which radiocomplexed drugs cleared from blood very quickly and then a slow phase in which they came out slowly (Fig. 1). 7 Biodistribution and scintigraphy All the compounds were evaluated for their capability of targeting the infection site compared to 99mTc-Ciprofloxacin. Biodistribution studies in mice were carried out in experimentallyinduced infection in the left thigh using Staphylococcus aureus. Both thighs of the mice were dissected and counted and the ratio of bacterial-infected thigh/contralateral thigh was then evaluated. The tissue uptake of 99mTc-Ciprofloxacin and the analogues (injected dose per gram of mice) was also determined for blood, heart, lungs, liver, spleen, kidney, stomach, intestine, normal muscle and infected muscle. 99m Tc-Ciprofloxacin–DTPA (99mTc-compound 9) showed the best infection vs. normal tissue ratio (3.35 : 1) better than the 99mTc-Ciprofloxacin (3.14 : 1) while that shown by 99m Tc-Ciprofloxacin–TETA (99mTc-compound 5) was (3.20 : 1). The least ratio was observed in the case of 99m Tc-Ciprofloxacin–DTPA-linker (99mTc-compound 8), 2.28 : 1. These results indicate the suitability of the synthesized fluoroquinolone analogues for infection imaging. As shown in Table 1, the radiolabeling efficiency of 99mTc- Ciprofloxacin

Fig. 1 Blood kinetics of the fluoroquinolone derivatives.

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0.02 0.12 0.31 0.09 0.44 0.48 1.35 0.47 0.03 0.03 0.09 0.02 0.14 0.15 0.59 0.14 0.17 0.03 0.15 0.03 0.39 0.47 0.47 0.30 0.06 0.08 0.13 0.05 0.15 0.36 0.53 0.17 0.79 0.44 0.16 1.21 4.27 5.34 2.59 4.81 0.25 0.15 0.04 0.23 1.22 1.02 0.45 0.78 1.11 1.06 0.12 0.26 6.55 7.58 1.95 1.90 0.19 0.10 0.32 0.07 0.75 0.85 1.04 0.41 0.18 0.09 0.22 0.18 0.61 0.24 0.58 0.47 0.20 0.13 0.13 0.23 3.27 1.91 3.02 2.20 1 1 1 1 : : : : 3.14 3.20 2.28 3.35 95.60% 93.89% 95.30% 92.90% 0.27 0.50 0.42 0.54

analogues synthesized in our work had similar profiles to that of 99mTc-Ciprofloxacin. Similar results were obtained in scintigraphic studies performed in the mouse model. Images taken at 1 h and 3 h using synthesized fluoroquinolone analogues as infection imaging agents showed the infection quite distinctly in the case of 99mTc-compound 8 and 99mTc-compound 9, and was comparable to 99mTc-Ciprofloxacin taken as a positive control. In the case of 99mTc-compound 5 the site of infection was not very distinct (Fig. 2). Activity accumulation in the liver & kidneys shows that the major route of excretion is hepatobiliary and renal. Low uptake of activity in the stomach is suggestive of in vivo stability of the radiolabeled complexes. The results indicate that the derivatization of the carboxyl group of Ciprofloxacin by the metal chelating group DTPA through a linker does not alter the radiochemical efficiency of the diagnostic formulation. Moreover, the better efficiency of the N-substituted Ciprofloxacin analogue indicates that the derivatization at nitrogen does not affect the diagnostic potential of the radiopharmaceutical. All the analogues synthesized in the present work can be further evaluated for their diagnostic efficacy in higher animals.

Tc-Compound Tc-Compound 99m Tc-Compound 99m Tc-Compound 99m

99m

Compound no.

1 5 8 9

99.54% 95.86% 97.98% 98.48%

Ratio of Biodistribution data of 99mTc-labeled Ciprofloxacin and its analogues in infected mice 4 h after injection. Each value is the mean SD of five infected vs. mice and expressed as percent injected dose per gram tissue (organ). normal Infected thigh Blood Heart Lungs Liver Spleen Kidney Stomach Intestine Muscle portion muscle % stability at 4 h in % radiolabeling saline efficiency solution

Table 1 Comparative results of biodistribution of Ciprofloxacin with its analogues in infected mice

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Fig. 2 Scintigraphic images in mice after 3 hours. Arrows indicate the site of infection. (a) 99mTc-Ciprofloxacin (b) 99mTc-Ciprofloxacin– DTPA. (c) 99mTc-Ciprofloxacin–TETA (d) 99mTc-Ciprofloxacin– DTPA-linker.


D. Discussion The major challenge for researchers designing radiopharmaceuticals to be used in diagnostic nuclear medicine is creating agents that will: 1. go to the target organ quickly, 2. go there as a high percentage of the injected radiation and Metallomics, 2009, 1, 409–417 | 415

3. clear from the body quickly once the test is completed. For most applications in diagnostic nuclear medicine the use of the isotope 99mTc is preferred. The idea of radiolabeling Ciprofloxacin was to preserve its capacity to bind bacteria and thereby enable the compound to specifically target those microorganisms. Specific binding of Ciprofloxacin to infecting microorganisms paves the way for radiological detection of infection sites. The objective of the present study was to develop novel radiopharmaceuticals based on the chemical structure of Ciprofloxacin and to compare their ability in comparison to Ciprofloxacin. The process of chelation was to be done keeping the basic structure of Ciprofloxacin intact providing an arm to chelate with 99mTc. Earlier pharmaceuticals involve direct chelation of 99mTc with Ciprofloxacin chelating through the carboxyl function combined with the keto function.9–12 The present research work involved synthesis of 99mTc-Ciprofloxacin analogues for imaging of infection while keeping in mind the following two important aspects: 1. To retain the structural integrity of the Ciprofloxacin molecule so that it does not lose its efficacy to bind to the target due to conformational changes. 2. To develop better radiopharmaceuticals based on the chemical properties of 99mTc-Ciprofloxacin by synthesizing new analogues. An ideal radiopharmaceutical should have a radiolabeling efficiency of more than 95%. The labeling efficiency of all of our compounds under investigation was more than this limit, indicating that the labeling efficiency of these radiopharmaceuticals was as good as other standard radiopharmaceuticals. Ideally the percentage of colloids present in the radiopharmaceutical formulation should be r0.5%. It was within limits in the case of 99mTc-Ciprofloxacin and 99m Tc-Ciprofloxacin–DTPA (99mTc-9), while the percentage of colloids present was a little more than the ideal limit in 99m Tc-Ciprofloxacin–DTPA-linker (99mTc-8). But in the case of 99mTc-Ciprofloxacin–TETA (99mTc-5) the percentage of colloids was certainly higher i.e. between 3 and 4%. The stability of radiolabeled compounds is one of the major problems in formulating radiopharmaceuticals. It must be stable both in vitro and in vivo. Temperature, pH, and light affect the stability of many compounds and the optimal range of these physicochemical conditions must be established for the preparation and storage of labeled compounds. In vivo breakdown of a radiopharmaceutical results in undesirable biodistribution of radioactivity. The complexes formed by chelation of 99mTc with our synthesized fluoroquinolone analogues were very strong complexes as suggested by in vitro serum stability. The biodistribution patterns observed in 99mTc-Ciprofloxacin and its analogues were high uptake in the kidneys with excretion to the urinary bladder, moderate to high uptake in the liver and spleen and blood pool activity. These results were in concordance with work done on 99m Tc-Ciprofloxacin.13 Following intravenous injection, fluoroquinolone derivatives were widely distributed in the body and were excreted via the kidneys. The radiopharmaceuticals bind to the bacteria present at the site of infection, which results in the retention of the agent at sites of active bacterial infection. The biodistribution data 416 | Metallomics, 2009, 1, 409–417

demonstrated that Staphylococcus aureus bacteria in animal infection models took up our radiopharmaceutical formulations, confirming our hypothesis that 99mTc-fluoroquinolones can work as diagnostic agents for targeted delivery in bacterial infections.

E. Conclusion The achievement of the present research work was the development of specific radiopharmaceuticals i.e. 99m Tc-fluoroquinolones for detection and localization of sites of bacterial infections in parts of the body. The observations extend the scope of 99mTc-fluoroquinolone radiopharmaceuticals for infection imaging/scintigraphy. These newly developed radiopharmaceuticals are safe & cost-effective. Our research work relates to diagnostic imaging of infections with metallic radionuclides. The parent fluoroquinolone molecule Ciprofloxacin, selected by us, is a potent broad-spectrum antibiotic and is active against most Gram-positive and Gram-negative bacteria. The pharmacological properties of antimicrobial drugs have been exploited for diagnostic use. The complex formed by chelation of 99mTc with these parent antibiotic molecules and their derivatives synthesized by us were strong as indicated by in vitro serum stability and in vivo biodistribution experiments. The in vitro and in vivo study results have shown satisfactory radiolabeling of fluoroquinolone derivatives with 99mTc, sufficiently stable up to 24 hours and have both hepatobiliary and renal routes of excretion. The biodistribution & scintigraphy studies gave evidence that 99mTc-fluoroquinolone based radiopharmaceuticals prepared in the present work have the ability to specifically bind to infecting microorganisms at the infectious foci. This is of potential clinical significance not only for correct diagnosis but also for the determination of the appropriate duration of antimicrobial treatment. Radiolabeled antimicrobial derivatives represent a novel approach to the diagnosis of deep-seated infection. The availability of commercial preparations, subject to rigorous quality control, will be an important component in the development of novel imaging agents for use in clinical practice.

Acknowledgements This work is supported by DRDO, Ministry of Defence, Government of India. One of the authors, Saurabh Dahiya, is also thankful to the Indian Council for Medical Research for providing a senior research fellowship.

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