2.1.

Fluorocoxib D

Fluorocoxib D was synthesized as previously described by Uddin.³⁹ The chemical structures of indomethacin, 5-carboxy-rhodamine, and fluorocoxib D along with their potencies as COX-inhibitors are shown in Fig. 1 and Table 1.

Fig. 1

Chemical structure of (a) indomethacin, (b) 5-carboxy-X-rhodamine (5-ROX), and (c) fluorocoxib D.

Table 1

Indomethacin, fluorocoxib A, and fluorocoxib D inhibit COX-1 and COX-2 activities.

In our in vivo experiments, fluorocoxib D was dissolved in dimethyl sulfoxide/ethanol/propylene glycol/saline solution to final concentration of $5\mathrm{mg}/\mathrm{ml}$ under sterile conditions and filtered using a $0.1\text{-}\mu \mathrm{m}$ pore size filter.

2.2.

Antibodies and Other Reagents

Antibody for COX-2 was obtained from Cayman Chemical Corporation (Ann Arbor, Michigan), and antibody for Ki67 was purchased from Dako (Carpinteria, California). All other chemicals and reagents were purchased from Thermo Fisher Scientific (Pittsburgh, Pennsylvania), unless otherwise specified.

2.3.

Human Cancer Cell Line

Human 5637 bladder cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, Virginia). Human 5637 cancer cells were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum, 100 I.U. penicillin, and $100\mu \mathrm{g}/\mathrm{ml}$ streptomycin (complete media) and grown in an atmosphere of 5% ${\mathrm{CO}}_2$ at 37°C.

2.4.

Fluorocoxib D Uptake by Human 5637 Cancer Cells

Human 5637 cells were seeded on four-chamber slides (Nalge Nunc, Rochester, New York) and grown until reached 70% to 80% confluence. Then, the cells were pretreated with or without $10\mu \mathrm{M}$ celecoxib for 30 min followed by a treatment with 50 nM fluorocoxib D for an additional 30 min. After this treatment, cells were washed with PBS and incubated for additional 1 h in complete media to remove any unbound fluorocoxib D. Cells were then washed twice with PBS and fixed in 10% formalin for 15 min at room temperature. The nuclei of cells were stained with DAPI for 10 min, and slides were mounted using an aqueous mounting medium. Images of cells were captured by a Leica DMi8 fluorescent microscope with Hamamatsu Orca Flash 4.0LT Digital CMOS Camera (Leica Microsystems Inc., Chicago, Illinois) using Leica Application Suite X software (LASX, Leica).

2.5.

Research Healthy Dogs

The animal studies were performed in the accordance with the University of Tennessee Institutional Animal Care and Use Committee (UT IACUC)-approved protocols and in an accordance with NIH guidelines. Individually housed research healthy female beagle dogs (Covance Research Product) with weights of 6.7 to 10.3 kg were used to assess the safety and pharmacokinetic parameters of fluorocoxib D administrated a dose of $1\mathrm{mg}/\mathrm{kg}$ over 20 min intravenously (i.v.).

2.6.

Pilot Safety Study of Fluorocoxib D in Research Dogs

For single dose safety evaluation studies, we administrated fluorocoxib D at $1\mathrm{mg}/\mathrm{kg}$, i.v. using a preplaced cephalic or saphenous vein catheter over 20 min to six healthy research dogs following protocol as described previously.³⁸ Briefly, physical examinations for signs of potential drug toxicity were performed before, during, and daily for 3 days after fluorocoxib D administration. Physical assessment included auscultation of heart and lungs, abdominal palpation, body temperature, appetite, attitude, activity levels monitoring, and other health-related events. Hypersensitivity was evaluated by direct observation of each dog during treatment for clinical signs of an allergic reaction (facial swelling, flushing, urticaria, dyspnea, scratching, and changes of heart rates). Signs of adverse events after fluorocoxib D administration were monitored daily, including vomiting, diarrhea, depression, nausea, and increased salivation. Laboratory evaluations of blood and urine were carried out before and 3 days after fluorocoxib D administration to monitor signs of toxicity. Blood (up to 3 ml) was collected into ethylenediaminetetraacetic acid (EDTA) and heparin-treated tubes, and urine (up to 5 ml) was collected by cystocentesis or from the floor of the runs. The laboratory evaluation consisted of a complete blood count (CBC), serum chemistry profile, and complete urinalysis. The CBC evaluated total white blood cells (WBC) and hematocrit (HCT), in addition to absolute numbers of neutrophils, lymphocytes, monocytes, eosinophils, and platelets. The plasma chemistry profile evaluated blood urea nitrogen (BUN), creatinine, proteins (albumin, globulins, and total proteins), alanine aminotransferase (ALT), total bilirubin, glucose, and electrolytes. The complete urinalysis evaluated urine proteins, glucose, and ketones; a sediment examination and urine-specific gravity were validated as well for any possible signs of renal toxicity of fluorocoxib D. All laboratory tests were carried out at the Veterinary Medical Center of the University of Tennessee in Knoxville.

2.7.

Pharmacokinetics of Fluorocoxib D in Research Dogs

To determine the pharmacokinetic parameters of fluorocoxib D ($1\mathrm{mg}/\mathrm{kg}$, i.v.) in dogs, blood (2 to 3 ml) was collected from the jugular vein into EDTA-treated tubes at 0, 0.5, 1, 2, 4, 8, and 24 h after compound administration. The blood was centrifuged within 20 min of sample collection. Plasma was removed and stored at $-80°\mathrm{C}$ until further HPLC analysis.

2.8.

HPLC Analysis of Plasma

Levels of fluorocoxib D were determined in collected canine plasma samples using HPLC at Vanderbilt University (JU). The frozen samples were thawed and extracted with acetonitrile. The organic layer was collected, dried, and reconstituted in methanol and water. The unknown samples were quantitated against a 5-point standard curve, which was prepared by mixing known quantities of fluorocoxib D with commercial canine plasma (Sigma-Aldrich) and subjecting the resulting samples to liquid–liquid extraction and HPLC. Samples were analyzed using a reverse-phase column (C18, $5\times 0.2\mathrm{cm}$, Phenomenex) with gradient elution. The mobile phase component A was water, and B was acetonitrile, each containing 0.1% acetic acid. The gradient was 50% B to 90% B over 5 min, followed by a brief hold and return to initial conditions. The flow rate was $0.3\mathrm{ml}/\min$.

2.9.

Pharmacokinetic Parameters

The pharmacokinetic parameters for fluorocoxib D were estimated from the plasma concentration-time data by a nonlinear mixed effects approach, as implemented with Monolix 4.1.2 (Lixoft S.A.S., Orsay, France). This approach allowed analyzing data from all animals at the same time and was ideal for relatively sparse datasets like the one used in this study. The plasma clearance (Cl), representing the overall ability of the body to eliminate fluorocoxib D, was determined by scaling its elimination rate (amount per time) by the corresponding plasma concentration level. The volume of distribution (Vd) was defined as the ratio of the total amount of fluorocoxib D in the body to the blood plasma concentration. The Cl and Vd were estimated directly from the study population data. The peak concentration in plasma ($C_{\max }$) and the following pharmacokinetic parameters were obtained by averaging the corresponding post-hoc Bayesian estimates. The individual partial areas under the curve between times 0 and 8 h (${\mathrm{AUC}}_{0-8}$) and the ${\mathrm{AUC}}_{0-\infty }$ with extrapolation to infinity were calculated using the log-linear trapezoidal rule, as implemented with WinNonlin 5.1 (Pharsight, Mountain View, California). The mean residence time (MRT) value was determined as the ratio of the area under the first moment curve over ${\mathrm{AUC}}_{0-\infty }$. The elimination rate constant was determined as the slope obtained by linear regression of the terminal log-linear portion of the concentration versus time curve, and the elimination half-life ($t_{1/2}$) was then calculated. The plasma concentration graphs were generated using calculated averaged plasma concentrations of fluorocoxib D from six dogs after $1\mathrm{mg}/\mathrm{kg}$ i.v. of fluorocoxib D administration performed over 20 min.

2.10.

Rhinoscopy-Guided Biopsy Sample Collection from a Client-Owned Dog with HNC Mass

A client-owned dog diagnosed with naturally occurring cancer was enrolled in our study through the Veterinary Medical Center by the Internal Medicine Service to validate the selective uptake of fluorocoxib D in cancer cells. The owners signed a consent form for enrolling their dog for our proof-of principal study. A nine-year old female-spayed Golden Retriever dog weighing 35 kg was presented for an acute onset of unilateral right-sided epistaxis of 5-day duration. Previous medical history included a grade 2 soft tissue sarcoma with a high mitotic index that was completely excised from her ventral neck $\sim 2$ years prior with no signs of regrowth. Rhinoscopy and biopsy procedure in the dog was performed by board-certified veterinary internal specialists (SO and JS) in an accordance with a standard veterinary care and the UT IACUC-approved protocol. Fluorocoxib D was administrated i.v. $1\mathrm{mg}/\mathrm{kg}$ over 20 min using a catheter, followed by a half hour uptake before initiation of rhinoscopy procedure. Rhinoscopy was used to obtain biopsy samples guided by uptaken fluorocoxib D in the computed tomography (CT)-identified head and neck cancer (HNC) mass. Rhinoscopy and biopsy procedures in dog were performed under general anesthesia using a 2.7-mm, 30-deg, 18-cm rigid scope (Karl Storz Veterinary Endoscopy) attached to a TRICAM PDD as described previously³⁸ using a custom-designed filter system for detection of fluorocoxib D (${\lambda }_{\mathrm{em}}$ $645\pm 50\mathrm{nm}$). After complete recovery from the rhinoscopic examination and anesthesia in a quiet room, the client-owned dog was returned to its owners. The heat map images with adjusted hue saturation of fluorocoxib D signal (pink color) from normal tissue (upper three images) and HNC tumor (lower three images) obtained during rhinoscopy were generated using Adobe Photoshop CC2019 software (Adobe Acrobat) in order to better visualize the tumor and its margins. Tumor-to-normal tissue ratio (T/N ratio) of fluorocoxib D signal obtained from normal ($n=804$) and tumor regions of interest (ROI) ($n=866$) from plot profile images generated from ImageJ software (NIH).

2.11.

Computed Tomography in a Client-Owned Dog with HNC Mass

CT of head in the client-owned dog was performed using a multislice helical CT scanner (Philips Brilliance-40, Philips International B.V., Amsterdam, Netherlands). A submillimeter multislice dataset of the head was acquired, and images were reconstructed in 0.9- and 4-mm slice thickness utilizing bone and soft tissue algorithms. The acquisition was repeated following intravenous administration of nonionic iodinated contrast medium (Ioversol $350\mathrm{mg}/\mathrm{ml}$, $2.2\mathrm{mg}/\mathrm{kg}$ [$1\mathrm{mg}/\mathrm{lb}$], Optiray^® $350\mathrm{mg}/\mathrm{ml}$, Tyco Healthcare/Mallinckrodt, Milwaukee, Wisconsin, USA). Images were evaluated by a board-certified veterinary radiologist (SH).

2.12.

Immunohistochemistry

The biopsy samples obtained during rhinoscopy procedures in dogs were formalin-fixed and paraffin-embedded and sectioned at $7\text{-}\mu \mathrm{m}$ thin sections for histology and immunohistochemistry analysis for detection of COX-2 and Ki67 expression as described previously.³⁸ For the antigen retrieval, sodium citrate buffer (10 mM, pH 6.0) was used for unmasking COX-2 and Ki67 proteins. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide, and nonspecific binding of secondary antibody was eliminated by incubation with protein block buffer (Biogenex USA, San Ramon, California) for 30 min at room temperature. Slides were incubated with primary antibodies for COX-2 (1:500) or Ki67 (1:500) overnight at 4°C, followed by incubation with specific secondary antibodies, streptavidin/biotin horseradish peroxidase complex (Biogenex), and visualized by DAB staining. Sections were lightly stained with Mayer’s hematoxylin. The tissue specimens were evaluated using a Leitz DMRB microscope. The images were captured by a DP73 camera attached to microscope using CellSens Standard software (Olympus, Pittsburgh, Pennsylvania).

3.1.

Safety of Administration of Fluorocoxib D to Research Dogs

To assess the safety of a single dose of fluorocoxib D, six female healthy research dogs were administered with fluorocoxib D ($1\mathrm{mg}/\mathrm{kg}$, i.v., over 20 min). A physical examination, CBC (Table 2), serum chemistry profile (Table 3), and complete urinalysis (Table 4) confirmed the clinically healthy status of the research dogs at baseline and 3 days after administration of fluorocoxib D. Of note, there were a few parameters that were outside of the normal laboratory reference ranges. Four dogs (dog #1, #3, #4, and #6) had low normal or mild hypoalbuminemia before and after administration of fluorocoxib D; the hypoalbuminemia remained stable in these dogs before and after fluorocoxib D administration (Table 3). One dog (dog #6) developed mild neutropenia 3 days after fluorocoxib D administration but remained afebrile and clinically normal; the cause of neutropenia in this dog was uncertain. The finding of hypoalbuminemia and mild hypocalcemia may reflect a chronic subclinical condition in these dogs. The mild hypernatremia (dog #6, Table 3) likely reflects subclinical dehydration given the well-concentrated urine (urine specific gravity 1.044, Table 4). The trace protein on urine dipstick is likely because the urine is well-concentrated. Three days after fluorocoxib D administration, two dogs (dogs #4 and #6) had bacteriuria (Table 4). Given the absence of pyuria, the bacteriuria could be iatrogenic or represent asymptomatic bacteriuria. The finding of bacteriuria is not thought to be relevant to this acute toxicity study. Adverse events including diarrhea, vomiting, nausea, and loss of appetite were not observed, although one dog exhibited increased salivation during the administration of fluorocoxib D over 20 min, most likely due to irritation from present DMSO as a one of the solvents of fluorocoxib D. Overall, the data indicate that single-dose administration of fluorocoxib D used at the dose of $1\mathrm{mg}/\mathrm{kg}$ was a safe dose for dogs.

Table 2

CBC analysis of blood before and 3 days after a single dose of fluorocoxib D (1 mg/kg, i.v.) in six healthy research dogs.

Table 3

Chemistry panel of serum before and 3 days after a single dose of fluorocoxib D (i.v., 1 mg/kg) in 6 healthy research dogs.

Table 4

Analysis of urine samples before and 3 days after a single dose of fluorocoxib D (1 mg/kg, i.v.) in six healthy research dogs.

3.2.

Pharmacokinetic Characteristics of Fluorocoxib D in Dogs

Pharmacokinetic parameters for fluorocoxib D were evaluated following i.v. administration of $1\mathrm{mg}/\mathrm{kg}$ over 20 min. Peak concentration of fluorocoxib D ($114.8\pm 50.5\mathrm{ng}/\mathrm{ml}$) was detected in plasma collected at 0.5 h after administration using a HPLC analysis as shown in Fig. 2. No fluorocoxib D was detected in plasma after 4 h following administration. The calculated pharmacokinetic parameters were shown as mean ± standard deviations (S.D.) in Table 5.

Fig. 2

The time course of fluorocoxib D plasma concentrations over 24 h of fluorocoxib D following administration by HPLC analysis. The data represent the mean plasma concentrations of fluorocoxib D (ng/ml) at specific time points following $1\mathrm{mg}/\mathrm{kg}$ i.v. administration over 20 min collected from research dogs ($n=6$) and validated by HPLC analysis.

Table 5

Pharmacokinetic parameters (mean ± S.D.) of fluorocoxib D determined after i.v. 1  mg/kg administration in six research dogs.

3.3.

Specific Uptake of Fluorocoxib D in COX-2-Expressing 5637 Cells In Vitro

To validate the specificity of fluorocoxib D uptake by human 5637 cancer cells, cells were pretreated with or without $10\mu \mathrm{M}$ celecoxib, a COX-2 selective inhibitor for 30 min, followed by 50-nM fluorocoxib D treatment for additional 30 min. Selective uptake of fluorocoxib D (red color) was detected in COX-2-positive 5637 cancer cells as shown in Fig. 3(a). Celecoxib prevented binding of fluorocoxib D to COX-2 enzyme in 5637 cancer cells as shown in Fig. 3(b). Nuclei of 5637 cells were counterstained with DAPI (blue) staining.

Fig. 3

Specific uptake of fluorocoxib D by human bladder cancer 5637 cells in vitro. (a) 5637 cells were treated with 50 nM fluorocoxib D for 30 min. (b) 5637 cells were pretreated with $10\mu \mathrm{M}$ celecoxib for 30 min followed by 50 nM fluorocoxib D treatment for 30 min. After treatment, cells were washed three times with PBS, incubated for additional 1 h with fresh complete media, fixed and counterstained with DAPI (blue). $40\times$ objective, scale bar $25\mu \mathrm{m}$. FD, fluorocoxib D; Cel, Celecoxib.

3.4.

Specific Uptake of Fluorocoxib D in COX-2-Expressing Cancer Cells In Vivo in a Dog During Rhinoscopy-Guided Biopsy Procedure

To validate the specific uptake of fluorocoxib D by COX-2-expressing cancer cells, we used a dog with naturally occurring and CT-identified HNC mass as a model of human cancer. In a dog CT revealed an ill-defined, lobular, mostly soft tissue attenuating, and mildly heterogeneous mass occupying almost the entire mid to caudal right nasal passage and resulting in extensive turbinate destruction as shown in Fig. 4(a). There was also evidence of early lysis and multifocal thinning of the nasal septum. The mass extended from the level of the right maxillary first premolar tooth to the level of the right fourth maxillary premolar tooth and filled the entire right maxillary recess. The mass displayed moderate heterogeneous contrast enhancement, with multiple rounded hypoattenuating regions with rim enhancement. The mass was visualized during rhinoscopy and directed biopsies were collected [Fig. 4(b)]. Specific uptake of fluorocoxib D was detected in HNC during rhinoscopy procedure in a dog as shown in Fig. 4(b) using white (middle panel) and fluorescence light exposures (right panel, red color). Histopathology analysis using H&E staining confirmed presence of HNC mass as a canine chondrosarcoma [Fig. 4(c), left panel]. The expression of COX-2 protein was confirmed in obtained HNC biopsy samples as shown in Fig. 4(c) (right panel, brown staining). The presence of Ki67-positive cells of HNC supported the evidence of proliferating cancer cells [Fig. 4(c), middle panel, brown staining]. T/N ratio of fluorocoxib D by canine HNC tumor was validated by ImageJ and Adobe PhotoShop CC2019 software as shown in Fig. 5. The representative heat map images of fluorocoxib D signal (pink color) from normal tissue (upper three images) and HNC tumor (lower three images) obtained during rhinoscopy using Adobe Photoshop CC2019 software (Adobe Acrobat) were shown in Fig. 5(a). The representative plot profile images of normal (left panel) and HNC tumor tissues using ImageJ software (NIH) were shown in Fig. 5(b). The averaged T/N ratio of fluorocoxib D signal obtained from normal ($n=804$) and tumor ROI ($n=866$) from plot profile images was in an average $3.7\pm 0.9$ as was shown in Fig. 5(c).

Fig. 4

Fluorocoxib D uptake by canine HNC tumor during rhinoscopy in vivo. (a) Transverse CT images of the nose in a 9-year-old Golden Retriever displayed in a bone window (left panel; window center 600 HU, window width 2600 HU) and in a soft tissue window (window center 50 HU, window width 350 HU) before (middle panel) and after (right panel) intravenous contrast medium administration. HNC mass (asterisk) is associated with and fills nearly the entire right nasal cavity. There is extensive lysis of nasal turbinates and thinning of the nasal septum. On precontrast images, the mass is soft tissue attenuating and mildly heterogeneous (left and middle panels). Following contrast medium administration, there is heterogeneous contrast enhancement with multiple round hypoattenuating regions with rim enhancement, suggesting hemorrhage or necrosis (right panel). (b) Planning of insertion of rigid scope to nasal cavity before rhinoscopy procedure (left panel). A representative image of localized HNC mass (asterisk) by a white light (middle panel) and under fluorescence light (right panel) with specific uptake of fluorocoxib D (red color). (c) A representative histology images of H&E (left panel), Ki67 (middle panel), and COX-2 (right panel) protein expressions in HNC of chondrosarcoma cells (asterisk) by immunohistochemistry staining (brown color). $20\times$ objective, scale bar $50\mu \mathrm{m}$.

Fig. 5

T/N ratio of fluorocoxib D by canine HNC tumor. (a) Representative heat map images of fluorocoxib D signal (pink color) from normal tissue (upper three images) and HNC tumor (lower three images) obtained during rhinoscopy using Adobe Photoshop CC2019 software (Adobe Acrobat). (b) Representative plot profile images of normal (left panel) and HNC tumor tissues using ImageJ software (NIH). (c) T/N ratio of fluorocoxib D signal obtained from normal ($n=804$) and tumor ROI ($n=866$) from plot profile images obtained from ImageJ software.