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. 2026 Oct 7. Online ahead of print. doi: 10.1039/d6ra08252a

Development and characterization of [99mTc]Tc-lornoxicam as a potential COX-assisted radiopharmaceutical for inflammation imaging

Atifa Batool a, Tania Jabar b, Syeda Minahal Fatima c, Syed Muhammad Waji-Ul-Hassan d, Nayyar Rubab b, Hafiz Kush Naseeb Ahmad b, Mohamed Fawzy Ramadan e, Nouf Aldawood f, Tahani O Alkahtani g, Abdulaziz A Alshihri h, Nadeem Ahmad Lodhi i, Syed Ali Raza Naqvi a,✉
PMCID: PMC13643229  PMID: 42846100

Abstract

Inflammation is a protective biological response; however, prolonged or dysregulated inflammation is associated with different pathological disorders. Conventional imaging techniques primarily visualize structural changes, while molecular radiotracers strongly support the understanding of the functional and biochemical characterization of inflammatory processes. The development of targeted radiopharmaceuticals for inflammation imaging remains a significant challenge in nuclear medicine. Oxicam-class non-steroidal anti-inflammatory drugs, particularly those associated with cyclooxygenase (COX) inhibition, provide a rational platform to develop inflammation-targeted radiopharmaceuticals. Therefore, the objective of this study is the synthesis and biological evaluation of [99mTc]Tc-lornoxicam as a SPECT inflammation imaging radiopharmaceutical. The radiosynthesis of [99mTc]Tc-lornoxicam was performed to optimize the reaction conditions, such as pH, lornoxicam concentration, SnCl2·2H2O concentration, and reaction time, using 2 mCi of radioactivity at ambient reaction temperature. Radiochemical quality was evaluated using instant thin-layer chromatography quality control (ITLC-QC) analysis and high-performance liquid chromatography (HPLC), and the in vitro stability, serum protein binding, and octanol/water partition coefficient (log P) of radiotracers were also determined. In vivo biodistribution was evaluated in turpentine-induced sterile inflammation and E. coli-induced infection rat models. Furthermore, scintigraphic imaging of radiotracer localization was carried out using single-photon emission computed tomography (SPECT) in New Zealand white rabbits. Under optimized reaction conditions, 94% radiochemical yield (RCY) was recorded. The stability study showed 90% and 92% intact radiocomplexes at 2 h in serum and saline, respectively, which further degraded to 82% and 86% at 4 h, respectively. A protein binding of 64% and a log P value of 1.23 ± 0.03 were recorded. Biodistribution study revealed sustained uptake in inflammatory tissue, demonstrating T/NT ratios of 2.45, 2.33, 2.13, and 2.28 at 5 min, 30 min, 2 h, and 4 h post-injection (p.i), respectively. SPECT imaging showed greater uptake of [99mTc]Tc-lornoxicam in sterile inflammation compared to E. coli infection, while free 99mTc showed nonspecific distribution. [99mTc]Tc-lornoxicam demonstrated a high labeling yield, satisfactory stability, moderate lipophilicity, and preferential inflammation-associated localization. These findings support the potential of the radiotracer as a COX-targeted radiopharmaceutical for inflammation imaging, although further molecular investigations are warranted to establish its COX-specific targeting mechanism.

Inflammation is a protective response that can be exploited for diagnosis. [99mTc]Tc-lornoxicam binds COX enzymes on inflammatory cells, and its γ-emission enables imaging of COX-associated pathological disorders.graphic file with name d6ra08252a-ga.webp

1. Introduction

Inflammation is a hallmark of numerous pathological conditions, including autoimmune disorders, infections, and cancer. It is a critical part of the immune response that is initiated to protect the host from infection, tissue injury, toxic stimuli and other harmful insults. Normally, the inflammatory response is a protective and self-limiting process, which includes endothelial activation, increased vascular permeability, vasodilation, the release of inflammatory mediators, and immune-cell recruitment to eliminate the damaging stimulus and promote tissue repair. However, when this response becomes excessively persistent or dysregulated, inflammation can contribute to tissue damage and disease progression.1,2 Chronic inflammation is associated with the pathogenesis of a variety of pathological conditions, including cardiovascular diseases, autoimmune disorders, neurodegenerative diseases, metabolic disorders, infections, and cancer.3–5 Specifically, cyclooxygenase-2 (COX-2) is an important molecular mediator in the inflammatory response because it takes part in arachidonic acid metabolism through prostaglandin biosynthesis. Recent reports have highlighted COX-2 as an important molecular target.6–8 Early non-invasive and molecular-level detection of inflammation not only facilitates disease diagnosis but also helps to understand disease activity, treatment response and pathological progression.

Conventional imaging techniques, such as X-ray, computed tomography (CT) and magnetic resonance imaging (MRI), are highly valuable (∼100% sensitivity) in diagnosing morphological disorders;9 however, a confusing outcome is obtained when there is no morphological change in inflamed tissues.10 Molecular imaging procedures (nuclear medicine techniques) based on sensing molecular targets using radionuclide-labeled targeted vectors promisingly resolve the tissue-morphology-associated sensitivity limitation. It helps explain the functional information by targeting specific cellular, biochemical or molecular processes taking place in pathological tissues.3,11–13 Previously, different synthetic and biological molecules were labeled with different radionuclides for inflammation and infection imaging. Among the currently available radiopharmaceuticals, 18F-FDG is one of the most widely used agents for inflammation imaging because of its high sensitivity and established PET/CT clinical utility. However, its uptake reflects increased glucose metabolism; therefore, it lacks sufficient specificity to discriminate sterile inflammation from infection or malignancy.14,15 Similarly, 67Ga/68Ga-citrate and radiolabeled leukocytes provide valuable approaches for infection and inflammation imaging but rely on relatively nonspecific accumulation or cellular trafficking mechanisms.16,17

Although current inflammation imaging agents, specifically 18F-FDG, radiolabeled leukocytes and 67/68Ga based tracers, provide sensitive detection of inflammatory lesions, the major limitation associated with these agents is limited molecular specificity. These agents commonly visualize non-specific biological responses, such as increased metabolism or vascular changes, as well as leukocyte recruitment; consequently, they are unable to provide reliable discrimination among infection, sterile inflammation and malignancies. Furthermore, physiological background uptake, false-positive accumulation, delayed imaging, and, in some particular agents, complex preparation procedures further limit their clinical utility.15,18,19 Therefore, there is an unmet need to develop such a small-molecule radiopharmaceutical that can exploit inflammation-associated molecular targets and may provide a more mechanistically defined imaging strategy. Within this context, a cyclooxygenase (COX)-enzyme inhibitory small molecule can offer a potential route toward the target-associated visualization of inflammatory tissue. Lornoxicam (Fig. 1) is an oxicam-class non-steroidal anti-inflammatory drug (NSAID) that works by blocking COX enzymes to reduce pain and inflammation. It is a reversible inhibitor of COX-1 and COX-2 enzymes.20 It is absorbed quickly: peak blood levels occur about 25 minutes after an intramuscular injection. More favorably, it has a short plasma elimination half-life of 3–4 hours, which is shorter than those of most oxicam drugs.21

Fig. 1. Chemical structure of lornoxicam.

Fig. 1

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Therefore, lornoxicam is an attractive candidate to label with 99mTc for imaging and diagnosing inflammatory lesions through interaction with inflammation-associated molecular targets. In this study, we present the radiolabeling of lornoxicam, offering a potential route toward the target-associated visualization of inflammatory tissue while retaining the practical advantages of 99mTc-based SPECT imaging.

2. Experimental

2.1. Chemicals

All the chemicals used in this study were of analytical grade and used without further purification. Lornoxicam was selected as the pharmacologically active ligand for radiolabeling, while sodium pertechnetate (Na99mTcO4) was freshly eluted from a 99Mo/99mTc PAKGEN (Moli) generator obtained from the Pakistan Institute of Nuclear Science and Technology (PINSTECH), Islamabad, Pakistan. During the radiolabeling procedure, a reducing agent (stannous chloride (SnCl2·2H2O)), stabilizing agents, a suitable buffer, and other auxiliary reagents were also used in their standard analytical-grade form. HPLC-grade methyl ethyl ketone (MEK), ethanol, and acetonitrile were purchased from Sigma-Aldrich for chromatographic analysis. For the partition coefficient study, n-octanol was purchased from Sigma-Aldrich (Germany), while for protein binding and stability investigation studies, required biological media/reagents were purchased from Solarbio Life Sciences and Oxoid (Thermo Fisher Scientific), Pakistan. All the aqueous solutions were prepared in distilled or deionized water. All reagents were freshly prepared for the radiolabeling reaction or stored under best-practice conditions to avoid chemical degradation or contamination that could affect the experimental results. Further, radioactive handling and radiochemical experiments were performed under established radiation-safety protocols. 99mTc-labeled samples were handled in lead (Pb)-shielded containers, and calibrated gamma-counting equipment was used for radioactivity measurements. A radioactivity-detector-equipped HPLC system was used to determine the radiolabeling purity.

2.2. Bacterial strain and animals

E. coli bacterial strains were obtained from the Department of Biochemistry, Government College University Faisalabad (GCUF). The Department of Physiology, GCUF, provided albino white rats for the biodistribution investigation, and New Zealand white rabbits were obtained from the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, for the scintigraphy study. All experimental animals were housed in a stress-free, controlled environment and provided with easy, continuous access to water and food. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Government College University Faisalabad, and the experiments were approved (No. GCUF/ERC/16/03) by the Animal Ethics Committee of Government College University Faisalabad.

2.3. Radiosynthesis of [99mTc]Tc-lornoxicam

To obtain the highest RCY, [99mTc]Tc-lornoxicam was prepared using an optimized radiolabeling procedure. In all radiolabeling experiments, a uniform 99mTc radioactivity of 2 mCi was used; to optimize reaction conditions, reagent concentrations were varied systematically. During optimization, the concentration of lornoxicam was varied from 0.5 to 1.0 mg mL−1, and that of SnCl2·2H2O was varied from 50 to 300 µg mL; the reaction pH was investigated from 6 to 12.

For radiolabeling, a known quantity of lornoxicam and SnCl2·2H2O in 1 mL of aqueous solution was added to a clean glass vial; the pH of the solution was adjusted using a 0.1 N solution of HCL or NaOH, and 2 mCi of freshly eluted Na99mTcO4 was added to the reaction mixture. The reaction volume was diluted to 2 mL with saline solution and vortexed moderately to mix all ingredients. The vortexed mixture was incubated for 20 minutes to complete 99mTc labeling with lornoxicam. Finally, the optimized reaction conditions were set by comparing the radiolabeling results obtained through a variety of investigations using systematically varied reaction conditions.

2.4. Determination of [99mTc]Tc-lornoxicam, free pertechnetate and hydrolysed radioactive impurities

At the end of the reaction period, the primary assessment for the determination of the [99mTc]Tc-lornoxicam complex, free 99mTcO4− (pertechnetate), and hydrolyzed technetium (99mTcO2) was performed using instant thin-layer chromatography (ITLC). To accurately determine the migration behaviors of the reaction impurities (free pertechnetate and hydrolyzed technetium), two different mobile-phase systems were used. For the determination of free 99mTcO4−, MEK was utilized as the mobile phase. For the analysis, an aliquot (∼2 µL) of the reaction mixture was applied at the baseline of the ITLC strip, and the strip was developed in MEK. In this chromatographic system, free 99mTcO4− migrated with the solvent front, while [99mTc]Tc-lornoxicam and hydrolyzed/reduced technetium species remained near the baseline. The developed strip was then cut into appropriate sections, and the radioactivity counts were measured using a sodium iodide (NaI) well-type gamma(γ)-counter. Solvent front activity (Rf = 1) was treated as free pertechnetate fraction activity, and the baseline and near-baseline activities were treated as [99mTc]Tc-lornoxicam and colloidal technetium activities. Upon complete development, the chromatographic strip was removed, dried, and cut into small strips, each 1 cm wide. The accurate percentage of free 99mTcO4− was determined using eqn (1):

2.4. 1

For the determination of hydrolyzed colloidal technetium, normal saline (0.9% NaCl) was used as the mobile phase. In this system, when the ITLC strip was developed, [99mTc]Tc-lornoxicam and free pertechnetate migrated along the mobile phase, while hydrolyzed colloidal technetium remained at the origin. Therefore, activity measured at the origin was calculated as the colloidal fraction, and migrated activity was treated as the non-colloidal fraction ([99mTc]Tc-lornoxicam and free pertechnetate). The hydrolyzed colloidal technetium was then calculated using eqn (2):

2.4. 2

Finally, the percent yield of [99mTc]Tc-lornoxicam was determined by combining the results of eqn (1) and (2) using eqn (3):

RCP (%) = 100 − [Free 99mTcO4− (%) + Colloidal 99mTc (%)] 3

The graphical illustration of the complete ITLC-QC protocol is presented in Fig. 2.

Fig. 2. Graphical illustration of the ITLC-QC protocol for the determination of radiochemical purity, free pertechnetate, and hydrolyzed radioactive impurity.

Fig. 2

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2.5. High-performance liquid chromatography (HPLC) analysis

The quality control of the radiotracer was conducted using the Thermo Scientific Ultimate HPLC 3000 system, which was integrated with a UV-vis NaI radioactive detector. An XTerra RP18 column (5 µm, 4.6 mm × 100 mm) was used. A sample, approximately 200 µL, after filtering through a 0.4 micron filter, was loaded into the HPLC injection port for chromatographic analysis. A gradient elution system was used with two solvent systems: solvent A: 0.1% TFA (trifluoroacetic acid) in water; solvent B: 0.1% TFA in ACN (acetonitrile). The gradient elution system was as follows: 0.0 min: 100% A, 0% B; 30 min: 0% A, 100% B; at an interval of 3 min, the concentration of A decreased by 10%, and the concentration of B increased by 10%.

2.6. In vitro stability of [99mTc]Tc-lornoxicam

The post-labeling stability of the [99mTc]Tc-lornoxicam radiocomplex was examined for up to 4 hours at room temperature and physiological temperature in saline and freshly harvested human blood serum, respectively. For up to 200 minutes, the 99mTc-labeled lornoxicam saline solution was set at room temperature. At predefined time points, i.e., 0, 50, 100, and 200 min (starting with the completion of the radiolabeling reaction), a 2 µL aliquot of the incubated mixture was withdrawn and applied at the baseline of the ITLC strip for chromatographic analysis (details given in the previous section) to determine the intact radiochemical.

2.7. Stability of the radiocomplex in blood serum

The in vitro stability of the radiolabeled complex was evaluated in blood serum to determine whether the complex remained intact or underwent 99mTc dissociation, transchelation, or degradation. Fresh blood was collected from a healthy subject and allowed to clot. The clotted blood was centrifuged at 3000 rpm for 10 min to separate the serum. The supernatant serum fraction was collected in a vial and used immediately. Thereafter, 100 µL of the radiolabeled complex was added to 900 µL of fresh blood serum. The mixture was vortexed gently to allow radiochemical–blood serum interaction, followed by incubation under approximately physiological conditions (37 °C and 5% CO2 humid air). At different incubation time points (0, 1, 2 and 4 h), an aliquot of the radiochemical–serum mixture was withdrawn and subjected to ITLC analysis to evaluate radiochemical integrity. ITLC analysis was performed using the chromatographic system explained in the previous section to determine the [99mTc]Tc-lornoxicam, free 99mTcO4− and colloidal technetium. The obtained radioactivity distribution in the radiochemical–blood serum mixture analysis was compared with that of the control sample (incubated in normal saline), and then the percentage of intact radiotracer was calculated based on the fraction corresponding to the radiolabeled complex using eqn (4):

2.7. 4

2.8. Protein binding

To investigate the protein-binding capacity of [99mTc]Tc-lornoxicam, we followed the reported protocol.22 Briefly, 1.5 mL of freshly collected blood serum from a healthy human volunteer was mixed with 0.5 mL of radiotracer solution (2–3 mCi). After thorough mixing, the serum–radiotracer mixture was incubated for 1 hour at 37 °C to allow both components to interact under near-physiological conditions. After incubation, the serum protein was precipitated by adding an equal volume of 10% trichloroacetic acid (TCA) solution. The sample was then centrifuged at 3000 rpm for 10 min, yielding protein-containing precipitate and soluble supernatant fractions. The precipitates were further suspended in 5% TCA solution and re-separated by centrifugation. A well-type γ-counter was used to measure the radioactivity of the precipitated and supernatant fractions of the serum. The protein-bound radiotracer fraction was expressed against the total recovered radioactivity, and the percentage protein binding was calculated using eqn (5):

2.8. 5

where total recovered radioactivity means the total radioactivity measured in protein precipitate and supernatant fractions.

2.9. Partition coefficient (log P) measurements

To assess the potential biological distribution behavior of [99mTc]Tc-lornoxicam, the lipophilic/hydrophilic character of the radiotracer was determined by calculating the octanol/water partition coefficient (log P). This parameter indicates the radiotracer's relative affinity for lipid and aqueous phases. We measured the partition coefficient by following the reported protocol.23 Briefly, a biphasic solution was prepared by mixing 1 mL of n-octanol and 900 µL of distilled water, followed by adding 100 µL of the [99mTc]Tc-lornoxicam radiopharmaceutical. The mixture was vigorously vortexed to ensure the proper interaction of the radiotracer with both phases and to achieve equilibrium distribution. The mixture was then centrifuged at 3000 rpm for 5 min to achieve clear phase separation. After phase separation, 100 µL aliquots were carefully withdrawn from each phase. The radioactivity of the octanol and aqueous phases was measured independently using a NaI well-type γ-counter. The experiment was performed in triplicate (n = 3), and the results were expressed as mean ± standard deviation (SD). Apparent partition P was calculated through the ratio of radiotracer concentration in the octanol and aqueous phases, as shown in the following expression:2.9.

However, the logarithmic form of partition P is termed the partition coefficient (log P), which is widely used to demonstrate the relative lipophilicity of radiotracers.2.9.

2.10. Biodistribution study of [99mTc]Tc-lornoxicam

Inflammation and infection were induced in albino rat models, which were then used to investigate the biodistribution of [99mTc]Tc-lornoxicam following the reported procedure with a slight modification.23 The animals were randomly divided into twelve (12) groups, each group consisting of three (n = 3) animals per time point. Half of the animals were injected with pure turpentine oil to induce sterile inflammation, and the other half were injected with E. coli bacteria to induce bacterial infection 48 h before radiotracer/free 99mTcO4− administration. Successful inflammation and infection induction were confirmed by observable redness and swelling at turpentine-oil/bacterial injection site. On the day of the experiment, all animals received 100 µL (37 MBq) of [99mTc]Tc-lornoxicam radiopharmaceutical and 100 µL (37 MBq) of free 99mTcO4− intravenously via the tail vein. At predetermined time intervals (5 min, 30 min, 2 h and 4 h p.i), the animals were anesthetized with chloroform and then sacrificed to excise selected organs and tissues, including the stomach, small intestine, large intestine, liver, spleen, lungs, heart and kidneys. Further, infected, inflamed and contralateral normal thigh muscle tissues were collected for comparative biodistribution analysis. Organs and tissues were excised very carefully to ensure that no tissue other than the targeted tissue was removed. All excised tissue samples were gently rinsed with distilled water to remove surface-associated or loosely adherent radioactivity. The washed tissues were then carefully blotted with adsorbent material to remove surface water, weighed accurately, and transferred to individually labeled γ-counting tubes to record radioactivity associated with each organ/tissue. The total activity injected into the animal was measured using syringe counts. The tissue uptake was expressed in terms of percentage injected dose per gram of tissue (% ID per g) using the following equation:% ID per g = [Radioactivity in tissue sample/Total injected radioactivity] × 100/Tissue weight (g)

2.11. Scintigraphy study of [99mTc]Tc-lornoxicam

To assess the biodistribution and accumulation of [99mTc]Tc-lornoxicam in infected and inflamed thigh muscles, as well as in various organs, scintigraphy was performed using a double-headed Siemens gamma camera (Germany) connected to the Xeleris Workstation, version 4, following the protocol reported in the literature.24 New Zealand white rabbits weighing approximately 1.5–2 kg were used; the right thigh muscle was infected with 500 µL of 1 × 108 colony-forming units (CFU) of E. coli, and the left thigh muscle was inflamed with 500 µL of sterile turpentine oil. The animal model with the most severe infection and inflammation was selected to administer the radiotracer for scintigraphy. A 20 mg kg−1 intramuscular injection of diazepam was used to anesthetize the rabbits, and then 500 µL of [99mTc]Tc-lornoxicam (2 mCi) was injected into the inferior ear vein in a pyrogen-free atmosphere. To capture anterior and posterior whole-body images at 5 min, 30 min, 2 h, and 4 h p.i, the sedated animal was positioned supine on a flat, hard surface.

3. Results

3.1. Physical characteristics

After optimizing the radiolabeling reaction conditions, the [99mTc]Tc-lornoxicam formulation became a transparent, pale yellow, odorless solution. Further, no particulate debris, visible precipitate or suspended matter was observed during visual inspection, which preliminarily indicates that no appreciable insoluble material or gross aggregation developed under the optimized reaction conditions.

3.2. Effect of quality control parameters on labeling yield

To develop high and reproducible radiolabeling efficiency, it is necessary to systematically optimize different quality-control (QC) parameters for the reaction conditions. Key QC parameters, such as ligand concentration, reducing-agent concentration and reaction pH, directly affect the efficiency, chemical speciation and radionuclide stability of radiolabeled complex formation. Therefore, these parameters were evaluated to identify optimized conditions for the maximum incorporation of 99mTc into the ligand cage and the minimum formation of undesirable radioactive species.

3.2.1. Effect of reaction pH on RCY

The effect of reaction pH on [99mTc]Tc-lornoxicam was investigated systematically to identify the optimum pH condition for maximum radiochemical yield (RCY). For this, pH values of 1–12 were evaluated, and at each condition, the RCY of the obtained radiolabeled product was assessed through ITLC-based QC, as illustrated in Fig. 3A. Lornoxicam was found to be insoluble at pH 1–4 and partially soluble at pH 5–7. Therefore, due to insufficient ligand solubility at pH 1–7, reliable complex formation and reproducible radiolabeling assessment are not possible. Conversely, under basic conditions, the solubility of lornoxicam was good, providing a favorable environment for radiolabeling and complex formation. At pH 11, the maximum RCY of 94% was obtained.

Fig. 3. Study of the effect of pH (A), reducing agent concentration (B), ligand concentration (C), and reaction period (D) on radiochemical yield.

Fig. 3

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3.2.2. Effect of SnCl2·2H2O concentration on RCY

For the synthesis of 99mTc-based radiopharmaceuticals, before the radiolabeling reaction, the oxidation state of freshly eluted 99mTcO4− was reduced to an appropriate lower oxidation state so that the reduced technetium could form a stable coordination complex with the donor atoms of the ligand. First, an appropriate reducing agent, SnCl2·2H2O, was selected, and its concentration effect on [99mTc]Tc-lornoxicam radiosynthesis was evaluated by systematically investigating concentrations of 75, 100, 150, 200, 250, and 300 µg mL−1. At each reducing-agent concentration, RCY was determined through an ITLC-based QC study. Fig. 3B presents the impact of reducing agents on radiolabeling yield. The optimum reducing-agent concentration of 100 µg mL−1 at pH 11 afforded a maximum RCY of 94%.

3.2.3. Effect of lornoxicam concentration on RCY

Lornoxicam is the key component of the radiosynthesis, alongside the radionuclide. The effect of ligand concentration, similar to pH value and reducing-agent concentration, was also evaluated systematically, with concentrations ranging from 0.1 to 0.35 mg mL−1 in 0.05 mg mL−1 increments. To identify the optimum lornoxicam concentration, all other reaction conditions were kept constant while ligand concentration was varied. As shown in Fig. 3C, a gradual increase in ligand concentration improves radiolabeling efficiency and reaches 94% labeling efficiency at 0.25 mg mL−1. The result indicated that 0.25 mg mL−1 of lornoxicam was sufficient to form an effective complex with reduced technetium species under optimal reaction conditions.

3.2.4. Effect of reaction time on RCY

Adequate reaction time is a key parameter for effective, maximum complex formation between reduced technetium species and lornoxicam. Therefore, this parameter was evaluated by allowing the reaction for 5, 10, 15, 20 and 25 min at ambient temperature, keeping all other reaction conditions constant. Fig. 3D shows that a 5 min reaction time gives a maximum RCY of 76%, which increases with the increase in reaction time. The maximum RCY of 94% was noted when the reaction was allowed to proceed for 20 min at ambient temperature.

3.3. Optimized radiolabeling reaction conditions

Based on the results of all the optimization experiments, lornoxicam concentration of 0.25 mg mL−1, SnCl2·2H2O concentration of 100 µg mL−1, reaction pH of 11, 2 mCi of freshly eluted 99mTcO4− and a 20 min incubation period with periodic gentle shaking at ambient temperature were identified as optimal reaction conditions, affording 94% RCY. Repeated labeling experiments using these optimal conditions showed easy, smooth and stable preparation of radiopharmaceuticals with high RCY. The preparation route and the proposed structure of [99mTc]Tc-lornoxicam are shown in Scheme 1.

Scheme 1. Preparation route and speculative structure of [99mTc]Tc-lornoxicam.

Scheme 1

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3.4. HPLC analysis

Prior to HPLC analysis, the sample was filtered through a 0.45 µm membrane to eliminate the colloidal impurity. HPLC (Fig. 4) revealed that approximately 2% free 99mTc was eluted at 4.38 min, while 98% [99mTc]Tc-lornoxicam was eluted at 13.76 min. This also reflected that the colloidal 99mTc fraction in the optimized formulation was about 4%.

Fig. 4. HPLC analysis of [99mTc]Tc-lornoxicam.

Fig. 4

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3.5. In vitro radiocomplex stability studies

The prepared [99mTc]Tc-lornoxicam was evaluated for in vitro stability in physiological saline and blood serum media to assess the chemical/radiochemical integrity of the radiolabeled complex in aqueous and biological environments. The stability assessment was performed at predetermined incubation intervals, i.e. 1, 2, 3, and 4 h. Fig. 5 showed no significant decrease in the intact radiochemical fraction; after 2 h of incubation, approximately 92% intact radiochemical fraction was observed, while 90% was observed in the blood serum. However, after 4 h of incubation, 86% and 82% intact [99mTc]Tc-lornoxicam were observed in saline and serum media, respectively.

Fig. 5. Stability of the [99mTc]Tc-lornoxicam complex in physiological saline and blood serum media.

Fig. 5

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3.6. Protein binding

The in vitro protein-binding behavior of [99mTc]Tc-lornoxicam was assessed in freshly collected heparinized human blood to evaluate radiotracer association with blood plasma proteins in a biological environment. According to the protein-binding analysis, [99mTc]Tc-lornoxicam showed approximately 64% protein binding.

3.7. Partition coefficient factor study

The lipophilic character of [99mTc]Tc-lornoxicam was assessed by determining the partition coefficient (log P) value using an n-octanol/water system. In triplicate, individual log P values of 1.23, 1.21, and 1.26 were obtained, with an average of 1.23 ± 0.03. This indicated that [99mTc]Tc-lornoxicam exhibited a moderate lipophilic behavior, suggesting that the radiochemical had a greater affinity for the n-octanol phase than the aqueous phase. Further, the relatively small variation among triplicate measurements and low standard deviation (±0.03) supported method reproducibility and experimental consistency.

3.8. Biodistribution study

The in vivo biodistribution of [99mTc]Tc-lornoxicam was evaluated in chemically induced sterile inflammation and bacterial infection animal models. [99mTc]Tc-lornoxicam and free technetium were administered through the intravenous route, and the biodistribution in all subgroups was evaluated at 5 min, 30 min, 2 h and 4 h p.i. At each time point, radioactivity associated with normal organs, inflamed tissues, and bacterial infection was quantified using a well-type γ-counter. Fig. 6 shows the biodistribution results for all four subgroups. [99mTc]Tc-lornoxicam clearly demonstrated a time-dependent distribution pattern.

Fig. 6. Biodistribution of [99mTc]Tc-lornoxicam in an inflammation-induced rat model (A), free 99mTcO4− in an inflammation-induced rat model (B), [99mTc]Tc-lornoxicam in an E. coli bacterial infection-induced rat model (C), free 99mTcO4− in an E. coli bacterial infection-induced rat model (D), and inflammation to infection (T/NT) values at specified time points (E).

Fig. 6

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As shown in Fig. 6A, initially, at 5 min p.i, the inflamed tissue showed very slow uptake (2.67% ID per g), which sharply increased to 7.09% ID per g at 30 min, but from 30 min to 2 h, very slow uptake was noted, with a 0.12% ID per g increase in 90 min. However, at 4 h, the uptake was 5.76% ID per g; the uptake rate may be lower compared to washout activity. Among the normal organs investigated in the inflamed animal model, the kidney showed the highest radiotracer uptake. Within 5 min, the kidney showed 32% ID per g uptake, which increased to 46% ID per g after 25 min (30 min time point) and reached its maximum value (48% ID per g) at the 2 h time point. However, at the 4 h time point, it decreased to 41% ID per g. Other organs showed minimal radiotracer uptake, except the liver, which showed 8.84% ID per g at 30 min, subsequently decreasing to 3.11% ID per g at 4 h p.i. The heart also showed higher uptake to some extent compared to other normal organs. A similar biodistribution pattern was recorded in bacterial infection-induced animal models (Fig. 6C); however, the infected tissues (non-target) showed significantly less uptake of radioactivity than the inflamed tissues (target). The radiotracer showed a significantly high target-to-non-target (T/NT) value of 2.45, 2.33, 2.13 and 2.28 at 5 min, 30 min, 2 h and 4 h, respectively. The T/NT profile suggested measurable contrast between inflammatory tissue and non-target tissue, even at high imaging time points (Fig. 6E). Further, for control investigation, both inflamed and infected animal models were injected with free 99mTcO4− under similar intravenous injection conditions. The biodistribution pattern was recorded at 30 min and 2 h p.i. These time points showed the non-specific accumulation of free technetium in the inflamed (Fig. 6B) and infected (Fig. 6D) animal models. Both Inflamed and infected tissues showed very low uptake.

3.9. Scintigraphy study

The scintigraphic study was conducted with experimentally induced sterile inflammation and bacterial infection models to investigate the in vivo localization, target-site uptake and specificity of [99mTc]Tc-lornoxicam. The experimental group received [99mTc]Tc-lornoxicam intravenously, and the control group received an equivalent free 99mTc activity. SPECT images were collected at 5 min, 30 min, 2 h and 4 h p.i. The scintigraphic images of the experimental group are shown in Fig. 7, while those of the control group are shown in Fig. 8.

Fig. 7. Scintigraphic scan images of [99mTc]Tc-lornoxicam in rabbit models with inflammation (left thigh muscle) and E. coli infection (right thigh muscle) at 5 min (A), 30 min (B), 2 h (C), and 4 h (D) p.i.

Fig. 7

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Fig. 8. Scintigraphic scan images of free 99mTc in rabbit models with inflammation (left thigh muscle) and E. coli infection (right thigh muscle) at 5 min (A), 30 min (B), 2 h (C), and 4 h (D) p. i.

Fig. 8

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4. Discussion

Under optimized reaction conditions, a lornoxicam concentration of 0.25 mg mL−1, SnCl2·2H2O concentration of 100 µg mL−1, reaction pH of 11, 2 mCi of freshly eluted [99mTcO4]− and a 20 min incubation period with periodic gentle shaking at ambient temperature, lornoxicam could conjugate with reduced 99mTc to obtain a stable complex. The availability of technetium from a relatively inexpensive molybdenum generator, with its patient-compatible gamma photon energy (140 keV), which is ideal for SPECT imaging, has made 99mTc the workhorse of the nuclear imaging community. Compounds of technetium have been reported in oxidation states ranging from +7 to −1, with 99mTc in +5, +3, and +1 oxidation states most commonly utilized in nuclear medicine.25,26 However, one of the most important findings so far has been the unanticipated importance of the (+5) oxidation state. Technetium complexes in this state have been found to be numerous, readily prepared, and often kinetically stable in an aqueous solution. Most of these newly discovered coordination compounds are stabilized by oxo groups, including 99mTcO+3, trans-99mTcO2+, and 99mTcO3+4.27–29 The labeling of lornoxicam with 99mTc can be rationalized on the basis of the ligand's ionizable and electron-donating functionalities, particularly the conjugated 4-hydroxy/enolic moiety and adjacent 3-carboxamide carbonyl group. Technetium, in its freshly eluted aqueous [99mTcO4]− form, bears a +7 oxidation state, which is a relatively inert state for direct coordination; therefore, in the presence of Sn(ii), [99mTcO4]− is reduced to lower oxidation-state technetium species. Most notably, as reported in the literature, lornoxicam could conjugate with 99mTcO+3 core to obtain a stable complex in the form of 99mTc(L2) (L = bidentate ligand); a speculative complex structure is shown in Scheme 1. In the chemical structure of lornoxicam, the 4-hydroxy/enolic moiety is conjugated with the adjacent 3-carboxamide carbonyl group, which plays a key role as an electron donor.30 The characteristic enolic structure of the oxicam class exhibits keto–enol tautomerism, in which electron delocalization occurs between the 4-hydroxy/enolic moiety and the adjacent 3-carboxamide carbonyl group. The deprotonation of enolic-OH may generate negatively charged oxygen species under alkaline conditions, which increases the electron-donating capacity of oxygen and the chance to coordinate with reduced technetium. Further, the 3-carboxamide carbonyl group can provide electron density for coordination; thus, the 4-hydroxy/enol-carbonyl domain may establish a potential chelating environment. The literature documents the coordination of different monodentate and bidentate metal ions with oxicam; however, N and O donor sites have also been documented to make transition-metal complexes with lornoxicam.31

The enhanced labeling efficiency observed at alkaline pH may therefore be attributed, at least in part, to the increased deprotonation of the 4-hydroxy/enolic functionality, resulting in the greater availability of electron-rich oxygen for coordination with reduced 99mTc species. Along with this, the potential coordinated contribution of carbonyl oxygen and (possibly) pyridine nitrogen also plays a role in stabilizing the complex. Further, under highly alkaline conditions, the possibility of hydrolysis and colloidal species formation of reduced technetium was mitigated by establishing a balance between optimum pH and ligand deprotonation, 99mTc reduction, metal–ligand coordination, and 99mTc hydrolysis suppression.

The radiochemical yield of [99mTc]Tc-lornoxicam after each experiment was monitored with ITLC and radio-HPLC, widely applicable and appropriate analytical approaches for evaluating radiolabeling, radiochemical purity, and the formation of a distinct radioactive species. Direct spectroscopic characterization can provide additional structural information; however, the spectroscopic characterization of a 99mTc-labeled, water-soluble radiopharmaceutical is technically challenging because the radiocomplex is present at tracer-level concentration and cannot readily be isolated in a sufficient quantity for conventional spectroscopic analysis. Erfani et al. developed [99mTc]Tc-tricarbonyl-meloxicam with 98.1% ± 0.4% labeling yield and reported good stability in human serum and pronounced accumulation in inflamed muscle.32 This study supports the concept that oxicam-class NSAIDs can successfully be labeled with 99mTc for inflammation imaging. The literature also revealed another important 99mTc-labeled pharmaceutical compound, [99mTc]Tc-ibuprofen, as a diagnostic agent for inflammatory target sites. It showed a high RCY and was successfully evaluated for inflammation imaging.33 Our study demonstrated the good stability of [99mTc]Tc-lornoxicam both in sterile saline and serum media. At 2 h post-incubation, 92% intact radiochemical was recorded in the saline medium, while 90% intact radiochemical was recorded in the serum medium. At 4 h post-incubation, this percentage decreased to 86% and 82%, respectively. However, the percentage of intact radiochemical increased by passing the radiocomplex solution through a 0.45 µm membrane. The slight decrease in radiochemical stability observed in the serum compared to the saline may be attributed to interactions with serum proteins and endogenous ligands, which can promote ligand exchange or the transchelation of the radiometal complex.34 However, despite the 82% intact radiochemical at 4 h post-incubation, it is an acceptable stability profile. Comparatively, [99mTc]Tc-MTX (a radiopharmaceutical developed for COX-targeted inflammation imaging) showed 94.9% ± 1.2% plasma stability at 2 h and 85.8% ± 2.7% at 4 h post-incubation, which were in good agreement with our results. Protein binding is a key determinant of radiopharmaceutical pharmacokinetics, because it influences the circulating free fraction, vascular residence, tissue penetration, biodistribution and clearance.35. [99mTc]Tc-MTX showed 48.1% ± 1.9% protein binding at 2 h, which increased to 49% at 4 h. These values are less than our recorded value, i.e. 64%@1 h post-incubation of [99mTc]Tc-lornoxicam, which is most probably due to the molecular structure, charge distribution, lipophilicity, protein-binding sites and overall physicochemical characteristics of the radiocomplex. Thus, our complex can slightly prolong blood clearance, as compared to the [99mTc]Tc-MTX complex. Furthermore, 64% protein binding and a 1.23 partition coefficient (log P) value indicated that the radiocomplex possessed an appreciable interaction with the blood compartment, which could subsequently affect the biodistribution profile; however, the reported [99mTc]Tc-MTX radiopharmaceutical showed a log P value of −2.28, facilitating hydrophilicity.36 Therefore, the positive log P value of [99mTc]Tc-lornoxicam theoretically facilitates membrane interaction and tissue penetration. However, log P alone cannot reliably predict in vivo targeting or tissue uptake, as tissue distribution is influenced by ionization state, pH-dependent lipophilicity (log D), protein binding, membrane permeability, and tissue-specific interactions.37,38 Furthermore, protein binding, molecular charge, ionization, blood flow, vascular permeability and tissue-specific interactions are also important parameters that determine target tissue uptake efficiency.39

A detailed biodistribution study of [99mTc]Tc-lornoxicam was performed in the inflammation and infection models and compared with the biodistribution in the control groups of both models by injecting free 99mTcO4−. [99mTc]Tc-lornoxicam showed almost similar biodistribution profiles in normal organs both in inflammation-induced (subgroup A, target; Fig. 6A) and in infection-induced (subgroup C, non-target; Fig. 6C) rat models. Prominent uptake was observed in the kidneys at 5 min p.i (32% ID per g for subgroup A) (Fig. 6A), which subsequently increased to 46% ID per g at 30 min p.i. The pronounced renal uptake may reflect the predominant role of the kidneys in the filtration and excretion of the radiotracer, although tubular reabsorption or retention may also contribute to renal activity.40 After 2 h, a decrease in activity in different organs and an increase in kidney uptake supported the progressive clearance of activity through the renal route. Interestingly, at 4 h, continuous glomerular filtration and excretion through the bladder most probably decreased the kidney uptake (40% ID per g). This was because most of the radioactivity from the normal tissues and blood was washed out through the kidneys.

In the liver, 3.5% ID per g activity was recorded at 5 min, which increased to 8.5% ID per g at 30 min and subsequently decreased at later time points. This transient hepatic uptake may reflect systemic distribution, followed by hepatic processing and subsequent excretory clearance, consistent with the recognized hepatobiliary handling of several 99nTc-labelled radiopharmaceuticals.41 In the heart, early uptake was observed, which dominantly reflected partially circulating blood-pool activity, showing the clearance of activity from the heart at subsequent time points during redistribution.

Importantly, in the case of the inflammation-induced rat model, 2.67% ± 0.27% ID per g uptake in the inflammatory tissues was observed at 5 min p.i, which increased to 7.09% ± 1.12% ID per g at 30 min p.i. Interestingly, at the subsequent time point, a sustained uptake of 7.21% ± 1.51% ID per g at 2 h was observed, which slightly washed out to approximately 1.45% ID per g at 4 h. The persistent retention of the radiotracer at the target site is important for molecular imaging, as sustained lesion-associated activity relative to background clearance can improve target-to-background contrast and facilitate lesion visualization.42 Conversely, in subgroup C (Fig. 6C), the biodistribution pattern showed an uptake of 1.09% ± 0.27% ID per g in bacterial-infected tissues, which increased to 3.0% ± 0.80% ID per g at 30 min and 3.39% ± 1.21% ID per g at 2 h p.i. Interestingly, following the uptake pattern that was observed in inflammatory tissues, the uptake in infected tissues at 4 h was also decreased to 2.53% ± 0.79% ID per g. The observed uptake of [99mTc]Tc-lornoxicam at the bacterial infection site is unlikely to reflect direct bacterial targeting; however, it may be associated with the inflammatory response accompanying infection. The comparatively high uptake in chemically induced sterile inflammation may reflect a more pronounced local inflammatory response, potentially providing a significant accumulation of the radiotracer.43,44 Utilizing the uptake data at the inflammatory tissues and bacterial-infected tissue, the T/NT values were calculated to be 2.45, 2.33, 2.13, and 2.28 at 5 min, 30 min, 2 h and 4 h, respectively. In this study, at all the time points, the T/NT ratio remained >2, which demonstrates measurable contrast between the inflammatory tissues and non-target tissues. However, calculating the T/NT ratio using radioactivity uptake in the normal thigh muscle, rather than the bacterial-infected tissue, yields significantly higher values at all time points than those obtained by comparing radiotracer uptake in the inflammatory tissue with that in bacterial-infected tissue. This claim was in good agreement with the reported study, in which [99mTc]Tc-MTX uptake in an inflammation model and normal muscles was investigated, and inflammation-to-muscle ratios of 3.12% ± 0.003% ID per g at 2 h and 2.92% ± 0.05% ID per g at 24 h were reported. Our findings showed somewhat lower values, but this was mainly due to the different inflammatory models.36 These findings are also consistent with the biodistribution results of radiolabeled NSAID drugs, such as [99mTc]Tc-tricarbonyl-meloxicam, which showed the highest accumulation in inflamed muscle, with a T/NT ratio of 3.90 at 4 h p.i.32 This high value may be due to differences in ligand structure, technetium coordination chemistry (use of tricarbonyl chemistry), animal model, inflammatory stimulus, administered activity and biodistribution kinetics intervals. In our study, sustained uptake in inflammatory tissues further supports the emerging concept that radiolabeled oxicam derivatives can demonstrate preferential localization in inflammatory lesions.32,45,46 Furthermore, the interference of free 99mTc in the biodistribution was ruled out by administering free 99mTc in both the inflammation-induced and infection-induced models. As shown in the pattern of biodistribution (Fig. 6B and D), no uptake was observed in the inflammatory and infected tissues; however, non-specific accumulation was recorded in normal organs, which was not observed during the radiotracer biodistribution study. This indicates that in the biodistribution of [99mTc]Tc-lornoxicam, free 99mTc was not present, or the quantity was minimal, representing no more than 2% of free 99mTc, as determined by HPLC (Fig. 4).

Finally, the biodistribution study was visually corroborated through a SPECT imaging study. The scintigraphy investigation in the experimental model showed prominent renal involvement in the activity secretion process, as shown in Fig. 7A. At the beginning, no prominent activity was visualized in the bladder, but in later images (Fig. 7B and C), the kidney and bladder both showed high uptake, and finally, as shown in Fig. 7D, the bladder was filled with radioactivity. Higher uptake in the sterile inflammatory site as compared to the infected site was observed in the scintigraphic images. This is in good agreement with the biodistribution profile and also indicates the possibility that the localization of the radiotracer was primarily associated with the inflammation-associated process instead of direct bacterial interaction. Low but measurable uptake at the E. coli-infected site can be explained on the basis of an inflammatory response. During bacterial infection, inflammatory mediators, local hyperemia, increased vascular permeability, leukocyte recruitment and tissue remodeling processes may occur, which can increase radiotracer delivery and retention. This phenomenon is strongly supported by reported data; [99mTc]Tc-ciprofloxacin was initially developed as a bacterial infection-specific imaging agent, but in subsequent studies, it showed uptake in both sterile inflammation and bacterial infection.47 Later, Sadra et al. reported, with clinical data, that [99mTc]Tc-ciprofloxacin failed to reliably discriminate septic and aseptic osteoarticular disease; even with 100% reported sensitivity, it showed 37.5% specificity.48 Therefore, the low uptake in the E. coli-infected tissues is not due to bacterial localization but due to the infection-associated inflammatory process. The scintigraphy study using free 99mTc further strengthens the current interpretation. Free technetium could not demonstrate accumulation in the inflamed and infected tissues but showed nonspecific accumulation in different body parts. Furthermore, the administration of non-radioactive lornoxicam prior to [99mTc]Tc-lornoxicam resulted in markedly reduced tracer accumulation at both the inflammatory and bacterial-infection sites in the rabbit model. This reduction may be attributed to the competitive occupation of available lornoxicam-associated binding sites by the unlabeled drug, thereby limiting the subsequent localization of the radiolabeled analogue. Because lornoxicam is a potent COX-inhibitory NSAID, the observed reduction in [99mTc]Tc-lornoxicam accumulation provides preliminary evidence for a COX-associated component in the tissue localization of the radiotracer. However, this blocking response cannot by itself establish exclusive COX-2 specificity, as lornoxicam may interact with COX-1/COX-2 and other inflammation-associated pathways. Therefore, the present findings support a potentially COX-associated mechanism of [99mTc]Tc-lornoxicam localization, while direct COX-2 expression and mechanistic competition studies are being pursued as part of our ongoing radiopharmaceutical development program. Erfani et al. developed the [99mTc]Tc-tricarbonyl-meloxicam radiopharmaceutical and demonstrated its good COX-mediated ability to image inflammation. Therefore, our study is in good agreement with the findings of Erfani et al., and it presents a strong possibility that [99mTc]Tc-lornoxicam preferentially targets sterile inflammation due to its affinity for the COX-2 enzyme.

5. Conclusion

Taken together, this study demonstrated that [99mTc]Tc-lornoxicam could be prepared successfully with 94% RCY. The optimized radiolabeling conditions demonstrated satisfactory physicochemical and radiochemical characteristics. The radiocomplex showed reasonable stability in saline and serum, with protein binding of 64% and log P = 1.23 ± 0.03. The biodistribution study showed prominent renal involvement in the radioactivity secretion process and sustained uptake in the inflammatory tissues, with a T/NT ratio of >2 throughout the investigation period. The biodistribution profile obtained using rat models was augmented by SPECT imaging, in which turpentine-induced sterile inflammation showed higher [99mTc]Tc-lornoxicam accumulation than E. coli infection, while free 99mTcO4− showed non-specific accumulation. Non-radioactive lornoxicam treatment prior to [99mTc]Tc-lornoxicam administration showed markedly reduced accumulation at the inflammation site, which indicated the possibility of COX-targeted accumulation of the tracer at the inflammation site. Furthermore, published studies on radiolabeled meloxicam drugs supported the feasibility of oxicam-based radiotracers for inflammation imaging. Therefore, on the basis of the current results, [99mTc]Tc-lornoxicam demonstrated potential for COX-targeted inflammation imaging. Future studies will focus on COX-2-specific competition and expression analysis, including the correlation of tissue tracer uptake with COX-2 levels and ex vivo mechanistic validation. In parallel, [99mTc]Tc-lornoxicam kit formulation will be optimized for reproducibility, stability, and quality control, followed by extended preclinical safety, pharmacokinetic and dosimetric evaluation toward eventual clinical translation.

Author contributions

Conceptualization: S. A. R. N. and T. J.; data curation: A. B., S. M. W., N. A., and T. O. A.; formal analysis: N. R. and A. A. A.; funding acquisition: H. K. N. A. and M. F. R.; investigation: A. B. and N. A. L.; methodology: A. B., H. K. N. A. and T. O. A.; project administration: S. A. R. N. and S. M. F.; resources: H. K. N. A., M. F. R., T. O. A. and N. A. L.; software: S. M. F., N. R. and N. A.; supervision: S. A. R. N. and T. J.; validation: S. M. W., M. F. R. and A. A. A.; visualization: T. J., S. M. F., S. M. W. and H. K. N. A.; writing – original draft: S. A. R. N.; writing – review & editing: A. B., T. J., N. R., N. A., A. A. A. and N. A. L.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors are very grateful to the Higher Education Commission (HEC), Islamabad, Pakistan, for their support in this work. The authors also appreciate the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R367), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, and the Deanship of Research and Graduate Studies at King Khalid University for funding this work through the Large Research Project under grant number RGP2/484/46.

Data availability

All data generated in this study are included in this published article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated in this study are included in this published article.

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