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Study of feline oral squamous cell carcinoma: Potential target for cyclooxygenase inhibitor treatment
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Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 245–250 www.elsevier.com/locate/plefa
Study of feline oral squamous cell carcinoma: Potential target for cyclooxygenase inhibitor treatment Lisa DiBernardia,1, Monique Dore´b, John A. Davisc, Jane G. Owensd, Sulma I. Mohammede,f, Carolyn F. Guptilla, Deborah W. Knappa,f, a
Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026, USA b Department of Pathology and Microbiology, University of Montreal, Montreal, Quebec, Canada c Amgen, Seattle, WA, USA d Elanco Animal Health, Greenfield, IN, USA e Department of Veterinary Pathobiology, Purdue University, West Lafayette, USA f Purdue Cancer Center and Purdue Oncological Sciences Center, Purdue University, West Lafayette, USA Received 15 November 2006; accepted 26 January 2007
Abstract Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality. A potential target for OSCC treatment is cyclooxygenase-2 (cox-2). Pet cats with naturally occurring OSCC may offer the opportunity to study anticancer activity of cox inhibitors. Cox-2 expression in feline OSCC was determined by immunohistochemistry. High intensity cox-2 immunoreactivity was detected in 6 of 34 (18%) feline OSCC samples. Weak immunoreactivity was noted in 22 OSCCs and in epithelial cells from oral mucosa of clinically normal cats. Pharmacokinetics of a cox inhibitor (piroxicam, 0.3 mg/kg) were studied in carcinoma-bearing cats to confirm a dose for follow-up trials. The average peak serum piroxicam concentration (948 ng/ml, which inhibited cox-2 activity) and serum half-life (15.9 h) were similar to that in normal cats. These results provide information (cox-2 expression as an inclusion criteria, 0.3 mg/kg daily piroxicam) for the design of follow-up trials of cox inhibitor treatment in pet cats with OSCC. r 2007 Elsevier Ltd. All rights reserved.
1. Introduction More than 300,000 people are diagnosed with oral squamous cell carcinoma (OSCC) worldwide each year [1]. OSCC is an aggressive malignancy which is associated with high morbidity and mortality rates [1]. New targets for the prevention and treatment of this form of cancer are needed. One of the more recently
Corresponding author. Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN, 47907-2026, USA. Tel.: +765 494 1107; fax: +765 496 1108. E-mail address: [email protected] (D.W. Knapp). 1 Current address: Veterinary Specialists of South Florida, Cooper City, FL, USA.
0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.01.006
proposed, potential targets for OSCC prevention and treatment is cyclooxygenase-2 (cox-2) [1,2]. Cox-2 is thought to have an important role in cancer development and progression [2,3], and cox-2 has been reported to be overexpressed in more than 75% of human OSCC samples [4]. Cox-2 and cox-2 products have been postulated to be involved in several aspects of cancer development and progression including: (1) inhibition of tumor cell apoptosis, (2) increased angiogenesis, (3) increased cancer invasiveness, (4) enhancement of inflammation with recruitment of growth factors, (5) immunosuppression, and (6) conversion of pro-carcinogens to carcinogens [3]. Although studies in OSCC have been limited, studies in this and other forms of cancer have demonstrated that drugs which inhibit cox-2 have anticancer activity and may enhance the
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anticancer effects of chemotherapy and radiation therapy [5–11]. Cox inhibitors used in these studies have included nonselective cox inhibitors which block the activity of cox-1 and cox-2 as well as selective cox-2 inhibitors. Animal studies are integral in assessing the anticancer activity of cox inhibitors. The most relevant animals for studies in OSCC, however, have not been determined [12]. Pet cats with naturally occurring OSCC could potentially offer the opportunity to study the effects of cox inhibitors against OSCC. OSCC is relatively common in pet cats comprising 75% of oral cancer and 3–6% of all cancer in this species [13,14]. Feline OSCC is an aggressive, rapidly progressing malignancy that is not responsive to chemotherapy (similar to OSCC in humans). The mortality rate in cats with OSCC is 490%, and survival of cats with this cancer has been limited to 1–4 months in most studies [13,14]. Feline OSCC metastasizes to regional lymph nodes and distant sites in some cases, although most cats die from local disease prior to the development of clinically detectable metastases [13,14]. The incidence of OSCC has been reported to be 11–45/100,000 cats yearly [14]. Therefore, with the current pet cat population exceeding 65 million in the United States, it is expected that more than 25,000 pet cats develop OSCC yearly. Previous reports have suggested that cox-2 expression is lower in feline OSCC than in human OSCC [15,16]. Preliminary work in our laboratory, however, suggests that cox-2 expression may be greater in feline OSCC than previously thought. It would be feasible to conduct a study of cox inhibitor treatment in cats with OSCC. Studies by the Purdue Comparative Oncology Program and other programs have demonstrated the willingness of pet owners to enroll their pet cats and dogs with cancer in clinical trials that will potentially benefit the individual animal, other animals with that form of cancer, and possibly humans with a similar form of cancer [8,9,17]. A drug which inhibits cox-2 (the nonselective cox inhibitor, piroxicam) is already in use in pet cats [18,19]. Although piroxicam is not FDA approved for use in cats, the drug is being prescribed for cats with a variety of conditions, and is thought to be generally well tolerated [18,19]. The pharmacokinetics of piroxicam in normal cats have been reported [19,20]. Regression of cutaneous SCC in two cats and delay in progression of OSCC in two cats have been noted following piroxicam treatment (unpublished data, Knapp and DiBernardi). This information, collectively, provides justification and lays some of the ground work for a clinical trial of piroxicam in pet cats with OSCC. Before undertaking such a study, however, it was considered appropriate to first determine the extent to which the target for piroxicam treatment (cox-2) is expressed in feline OSCC and to confirm the dosage of piroxicam to be used. This study, therefore, was
conducted to assess: (1) cox-2 expression in feline OSCC, (2) the pharmacokinetics of piroxicam in carcinomabearing cats, and (3) the cox-2 inhibitory activity (measured as in vitro PGE2 release from peripheral blood monocytes) of piroxicam at the various concentrations which were recorded in carcinoma-bearing cats.
2. Materials and methods 2.1. Expression of cox-2 in feline OSCC archival tissues Thirty-four paraffin embedded archival tissue samples of feline OSCC and five samples of normal oral mucosa from non-tumor bearing cats were evaluated for cox-2 protein by immunohistochemistry (IHC). Tissue from a canine prostatic adenocarcinoma served as a positive control. A specific cox-2 antibody (MF243) was generously provided by Drs. Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Doval, Quebec, Canada). MF243 was raised in rabbits against ovine placental cox-2, and its selectivity for cox-2 has previously been characterized [21,22]. Fig. 1 demonstrates that this antibody also cross-reacts with feline cox-2 as it recognized a 72,000– 74,000 molecular weight doublet in a cell protein extract from cultured feline fibroblasts. Immunohistochemical staining was performed using the Vectastain ABC kit, as previously described [23]. Briefly, formalin-fixed tissues were paraffin-embedded, and 3 mm thick sections were prepared and deparaffinized through graded alcohol series. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum for 20 min at room temperature. Primary antibody diluted in PBS was applied (1:7500 dilution), and sections were incubated overnight at 4 1C. Control sections were incubated with PBS or with non-immune rabbit serum. After rinsing in PBS for 10 min, a biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) (1:222 dilution) was applied, and sections were incubated for 45 min at room temperature. Sections were washed in PBS for 10 min, and incubated with the avidin DH-biotinylated horseradish peroxidase H reagents for 45 min at room temperature. After PBS wash for 10 min, the reaction was revealed using diaminobenzidine tetrahydrochloride (DAB) as the peroxidase substrate. Sections were counterstained with Gill’s hematoxylin stain and mounted. The percentage of positively staining tumor or normal epithelial cells and the staining intensity were recorded in multiple fields. The staining intensity was categorized as; 0—no staining, 1—weak or equivocal staining, 2—moderate intensity staining or, 3—marked intensity staining.
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Fig. 1. Cross-reactivity of antibody MF243 for feline cox-2. A solubilized cell extract was prepared from cultured feline fibroblasts (obtained from ATCC) and analyzed by one-dimension SDS-PAGE gel and immunoblotting, as previously described [22]. Markers on the left indicate migration of molecular weight standards.
2.2. Pharmacokinetics of the cox inhibitor, piroxicam, in carcinoma-bearing cats Studies in cats were performed with the approval of the Purdue Animal Care and Use Committee. Eight carcinoma-bearing cats were admitted to PUVTH after acquiring informed owner consent. A jugular catheter was placed in each cat. Piroxicam (PCCA, Houston, TX) was given at a dose of 0.3 mg/kg orally. The powder was weighed, and encapsulated to achieve the desired dose for each individual cat. Blood samples were drawn at 0, 1, 2, 4, 12 and 24 h post piroxicam administration. Blood was collected and placed in a serum separator tube, centrifuged at 5000 rpm, at which time the serum was separated and frozen at 20 1C. Serum analysis was generously performed by Pfizer Global Research & Development Laboratories utilizing high performance liquid chromatography (HPLC) and mass spectroscopy. Following thawing and addition of an internal standard (CP-016,460), serum samples (100 ml) were extracted with 2 ml of ethyl acetate. The organic layers were removed and placed under a stream of nitrogen at 40 1C. The residues were reconstituted with 200 ml of an equal
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mixture of solvent A and B (see below). The samples were agitated, centrifuged and transferred to an autosampler apparatus. Analysis was performed on a SCIEX API 150 HPLC/MS system. The HPLC column effluent was directed into the source via an ion spray interface operated at 2000 eV and the mass spectrometer was operated in the negative ion mode. The HPLC system consisted of a Shimadzu SIL-10AD VP auto-sampler and an HP-1100 de-gasser and gradient pump. Chromatography was carried out on a Phenomenex Luna HPLC column with a binary mixture of solvent A (methanol) and solvent B (10 mM NH4OAc). A flow rate of 0.3 ml/min was used for all analyses. The mobile phase initially consisted of solvent A/solvent B (5/95) for 1 min. It was linearly programmed to achieve a mixture of solvent A/solvent B (95/5) over a 3 min period and operated isocratically for 3 min under these conditions. The mobile phase was then linearly programmed to return to the initial mixture of solvent A/solvent B (5/95) over a 1 min period and operated isocratically for 2 min. This gave a total run time of 10 min. Chromatographic results were recorded using Mass Chrom v. 1.1.1, and were integrated for quantification using MacQuan v. 1.6. The standard curve dynamic range was 10– 4000 ng/ml with a correlation coefficient of 0.985 using a regression analysis weighted 1/x2. These data were analyzed with a non-compartmental, extra-vascular pharmacokinetic model using WinNonlin v. 2.1. 2.3. Cox-2 inhibition in vitro The production of prostaglandin E2 (PGE2) in feline peripheral blood monocytes was determined as a measure of cox-2 activity [24]. Mononuclear cell isolation was performed as described by Guptill with minor modifications [25]. Plastic adherence was used to separate the nonadherent lymphocytes from the adherent monocytes. Monocytes were then treated with piroxicam concentrations of: 1 mM (0.33 mg/ml), 3 mM (1.0 mg/ml), 10 mM (3.33 mg/ml), or 30 mM (10 mg/ml) or piroxicam vehicle (control). Lipopolysaccharide (LPS 10 mg/ml) was added to each well to stimulate cox-2 activity. Following 18 h of incubation at 37 1C, the media was collected and stored at 80 1C until analyzed for PGE2 concentration. PGE2 concentrations were determined utilizing a Prostaglandin E Metabolite EIA kit (Cayman Chemical Company, Ann Arbor, MI) that measures the metabolites of PGE2. The manufacturer’s protocol was followed.
3. Results 3.1. Expression of cox-2 in feline OSCC archival tissues Immunohistochemistry (IHC) to detect cox-2 protein was performed on 34 samples of feline OSCC. Of these
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Fig. 2. Expression of cox-2 in feline normal oral mucosa and oral squamous cell carcinoma. Immunohistochemistry was performed on formalin-fixed sections with antibody MF243 (selective for cox-2). An avidin-biotin-peroxidase complex method with Gill’s hematoxylin counterstain was employed. Weak immunoreactivity (note pale brown staining) was noted in the upper layers of the normal oral mucosa (A). The majority of feline oral squamous cell carcinomas had weak cox-2 immunoreactivity (B), while some tumors demonstrated strong cox-2 immunoreactivity (note intense brown staining in C). Negative control sections (PBS alone) had no immunostaining (D).
Table 1 Serum pharmacokinetic parameters of piroxicam in cats following 0.3 mg/kg p.o. Serum pK parameters
Tmax (h) Cmax (ng/ml) AUCClast (ng h/ml) AUC (ng h/ml) AUC (% ext.) T1/2 (h)
Cat number #1
#2
#3
#4
#5
#6
#7
#8
2.0 1032.5 15217 24820 38.7 16.9
2.0 895.9 12257 15378 20.3 10.3
2.0 579.0 10268 27842 63.1 33.6
2.0 1345.1 16726 22005 24.0 11.4
2.50 905.5 13998 19936 29.8 13.3
1.0 937.8 11318 14165 20.1 9.8
2.0 1105.3 16830 25093 32.9 14.9
1.0 783.7 10376 16626 37.6 16.7
34 OSCC samples, 28 displayed some cox-2 immunoreactivity with intensity varying from weak (staining intensity 1) to strong (staining intensity 3) (Fig. 2B and C). The majority (22/28) of cox-2-positive tumors had weak immunostaining that appeared as diffuse cytoplasmic immunoreactivity in tumor cells (Fig. 2B). Six of the OSCC had strong cox-2 staining intensity in the cytoplasm of neoplastic keratinocytes (Fig. 2C). The percentage of tumor cells with cox-2 immunoreactivity ranged from 15% to 80%. Six OSCC tissues had no cox2 immunoreactivity. Weak cox-2 immunoreactivity was observed in epithelial cells in 4 of 5 samples of normal feline oral mucosa. The immunostaining was mostly localized to the upper layers of the mucosa (Fig. 2A).
Avg.
STD
1.81 948.1 13374 20733 33.3 15.9
0.5 225.9 2701 5031 14.0 7.7
3.2. Pharmacokinetics of piroxicam in carcinoma-bearing cats Pharmacokinetic data are summarized in Table 1. The average maximum plasma concentration of 948 ng/ml (0.948 mg/ml) occurred at approximately 2 h post oral piroxicam dosing for most cats. The average serum halflife of piroxicam was 15.9 h. 3.3. Cox inhibition in vitro Cox-2 activity, measured as monocyte production of PGE2, was studied in blood samples from five cats. The IC50 of piroxicam was less than 1 mM (0.33 mg/ml). Piroxicam at a concentration of 1 mM inhibited cox-2
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activity by 84.8710.7%. Piroxicam at a concentration of 3 mM (1.0 mg/ml) inhibited cox-2 activity by 9075.6%.
4. Discussion and conclusions In preparation for possible trials of cox inhibitor treatment in cats with OSCC, the extent of cox-2 expression was assessed in feline OSCC tissues. Cox-2 is thought to be an important target for cox inhibitor treatment of cancer. Most, although not all, studies implicate cox-2 as the major mechanistic target for the anticancer activity of these drugs in cancer patients [2,26–29]. High intensity immunoreactivity to cox-2 was noted in 18% of the feline OSCC samples studied with positive staining in 15–80% of tumor cells in these samples. Although weak immunoreactivity was noted in several other cases, the importance of this is not known, especially in lieu of the finding of weak immunoreactivity in normal keratinocytes in normal oral mucosa. In a previous report, cox-2 immunoreactivity was noted in 2 of 21 feline OSCC samples and was limited to weak to moderate intensity staining in 10% or less of cancer cells [16]. In a second report, cox-2 immunoreactivity was noted in 37 of 55 feline OSCC samples, but staining was present in o10% of cells and was only of mild to moderate intensity [15]. Although our study demonstrated more cox-2 immunoreactivity than previously reported for feline OSCC, it still appears that cox-2 expression in feline OSCC is lower than that in human OSCC where 475% of OSCC samples express cox-2. The cause for this difference in cox expression between species is not known. This does not mean that pet cats with OSCC should not be used for cox inhibitor studies, rather that cox-2 expression in the cancer cells be used as an inclusion criteria for cats to participate in such studies. Our group has previously reported the frequent cox-2 expression in naturally occurring canine OSCC, and dogs could also be considered as subjects for cox inhibitor studies [8,30]. Some of the attraction to studies in pet cats is that OSCC in the cat is more common, more devastating, and more lethal than that in dogs. OSCC in cats has been linked to tobacco smoke exposure (as in humans) [31]. Because of the grave prognosis for cats with OSCC and short survival (1–4 months), pet owners are eager for their cats to participate in treatment trials of potentially beneficial, relatively nontoxic therapy. Piroxicam was selected for study because this drug was already in use in pet cats with a variety of conditions [18], it has been generally well tolerated, and it has been associated with possible anticancer activity. Also, other cox inhibitors were not available for use in cats when this work began. The anticancer
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activity of piroxicam has not been formally studied in pet cats, but some evidence exists that this drug has anticancer activity in cats. Partial remission (450% reduction in tumor volume) of cutaneous SCC has been observed in two pet cats (unpublished data, Knapp and DiBernardi) receiving piroxicam (0.3 mg/kg daily per os). In two cats with locally advanced OSCC, a delay in cancer progression was noted after institution of piroxicam (0.3 mg/kg daily per os) for pain relief, and survival of the cats was longer than expected (unpublished data, Knapp and DiBernardi). In nine other cats with advanced OSCC receiving piroxicam, however, anticancer activity was not observed. Piroxicam pharmacokinetics were determined to help confirm the choice of piroxicam dose for follow up trials. The dosage of piroxicam (0.3 mg/kg daily per os) was selected because this dose has been used in cats, and this dose appears to be well tolerated. In addition, the pharmacokinetics of piroxicam given at this dose to normal non-cancer bearing cats have been previously reported [19,20]. Results in the normal cat were similar to our findings in carcinoma-bearing cats in this study. Although it was expected that carcinoma-bearing cats would metabolize piroxicam in a similar fashion to that of normal cats, it was appropriate to confirm this prior to larger studies of the anticancer activity. The half-life of piroxicam in the blood of carcinoma-bearing cats was estimated to be 15.9 h. The short duration of sample collection relative to the half-life of the drug resulted in all cats demonstrating an extrapolatedarea under the curve (AUC) greater than 20%. Thus, the serum half-life of 15.9 h in this study is considered an estimate. The average peak piroxicam concentration measured in carcinomabearing cats following a single dose of piroxicam (948 ng/ ml) was found to be sufficient to block cox-2 enzyme activity (the major target of cox inhibitor therapy). While it would be helpful to know the piroxicam concentrations achieved in the cancer tissues and the length of time piroxicam remains in cancer tissues, assays to perform such measurements were not available. The results of this study provide insight into the design of trials of cox inhibitor treatment in cats with OSCC. The pharmacokinetics work suggests that the dose of piroxicam in current use in cats (0.3 mg/kg) would be appropriate for use in a clinical trial. Inclusion criteria for the trial should include overexpression of cox-2 in cancer cells from that cat. Although this may only allow inclusion of approximately 20% of cats with OSCC, with the devastating nature of this cancer in pet cats, a potentially beneficial treatment option for 20% of affected cats would represent a notable improvement compared to current therapy [13,14]. Pet cats may benefit from cox inhibitor treatment trials while important information is being generated to define the potential application of this therapy approach for animals and humans.
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Acknowledgements The authors would like to thank Lisa Holeman, Debra Schlittler, Rose Thomas, Sarah Bradley, and Jane Stewart for their assistance with this work and manuscript preparation.
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Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 245–250 www.elsevier.com/locate/plefa
Study of feline oral squamous cell carcinoma: Potential target for cyclooxygenase inhibitor treatment Lisa DiBernardia,1, Monique Dore´b, John A. Davisc, Jane G. Owensd, Sulma I. Mohammede,f, Carolyn F. Guptilla, Deborah W. Knappa,f, a
Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026, USA b Department of Pathology and Microbiology, University of Montreal, Montreal, Quebec, Canada c Amgen, Seattle, WA, USA d Elanco Animal Health, Greenfield, IN, USA e Department of Veterinary Pathobiology, Purdue University, West Lafayette, USA f Purdue Cancer Center and Purdue Oncological Sciences Center, Purdue University, West Lafayette, USA Received 15 November 2006; accepted 26 January 2007
Abstract Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality. A potential target for OSCC treatment is cyclooxygenase-2 (cox-2). Pet cats with naturally occurring OSCC may offer the opportunity to study anticancer activity of cox inhibitors. Cox-2 expression in feline OSCC was determined by immunohistochemistry. High intensity cox-2 immunoreactivity was detected in 6 of 34 (18%) feline OSCC samples. Weak immunoreactivity was noted in 22 OSCCs and in epithelial cells from oral mucosa of clinically normal cats. Pharmacokinetics of a cox inhibitor (piroxicam, 0.3 mg/kg) were studied in carcinoma-bearing cats to confirm a dose for follow-up trials. The average peak serum piroxicam concentration (948 ng/ml, which inhibited cox-2 activity) and serum half-life (15.9 h) were similar to that in normal cats. These results provide information (cox-2 expression as an inclusion criteria, 0.3 mg/kg daily piroxicam) for the design of follow-up trials of cox inhibitor treatment in pet cats with OSCC. r 2007 Elsevier Ltd. All rights reserved.
1. Introduction More than 300,000 people are diagnosed with oral squamous cell carcinoma (OSCC) worldwide each year [1]. OSCC is an aggressive malignancy which is associated with high morbidity and mortality rates [1]. New targets for the prevention and treatment of this form of cancer are needed. One of the more recently
Corresponding author. Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN, 47907-2026, USA. Tel.: +765 494 1107; fax: +765 496 1108. E-mail address: [email protected] (D.W. Knapp). 1 Current address: Veterinary Specialists of South Florida, Cooper City, FL, USA.
0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.01.006
proposed, potential targets for OSCC prevention and treatment is cyclooxygenase-2 (cox-2) [1,2]. Cox-2 is thought to have an important role in cancer development and progression [2,3], and cox-2 has been reported to be overexpressed in more than 75% of human OSCC samples [4]. Cox-2 and cox-2 products have been postulated to be involved in several aspects of cancer development and progression including: (1) inhibition of tumor cell apoptosis, (2) increased angiogenesis, (3) increased cancer invasiveness, (4) enhancement of inflammation with recruitment of growth factors, (5) immunosuppression, and (6) conversion of pro-carcinogens to carcinogens [3]. Although studies in OSCC have been limited, studies in this and other forms of cancer have demonstrated that drugs which inhibit cox-2 have anticancer activity and may enhance the
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anticancer effects of chemotherapy and radiation therapy [5–11]. Cox inhibitors used in these studies have included nonselective cox inhibitors which block the activity of cox-1 and cox-2 as well as selective cox-2 inhibitors. Animal studies are integral in assessing the anticancer activity of cox inhibitors. The most relevant animals for studies in OSCC, however, have not been determined [12]. Pet cats with naturally occurring OSCC could potentially offer the opportunity to study the effects of cox inhibitors against OSCC. OSCC is relatively common in pet cats comprising 75% of oral cancer and 3–6% of all cancer in this species [13,14]. Feline OSCC is an aggressive, rapidly progressing malignancy that is not responsive to chemotherapy (similar to OSCC in humans). The mortality rate in cats with OSCC is 490%, and survival of cats with this cancer has been limited to 1–4 months in most studies [13,14]. Feline OSCC metastasizes to regional lymph nodes and distant sites in some cases, although most cats die from local disease prior to the development of clinically detectable metastases [13,14]. The incidence of OSCC has been reported to be 11–45/100,000 cats yearly [14]. Therefore, with the current pet cat population exceeding 65 million in the United States, it is expected that more than 25,000 pet cats develop OSCC yearly. Previous reports have suggested that cox-2 expression is lower in feline OSCC than in human OSCC [15,16]. Preliminary work in our laboratory, however, suggests that cox-2 expression may be greater in feline OSCC than previously thought. It would be feasible to conduct a study of cox inhibitor treatment in cats with OSCC. Studies by the Purdue Comparative Oncology Program and other programs have demonstrated the willingness of pet owners to enroll their pet cats and dogs with cancer in clinical trials that will potentially benefit the individual animal, other animals with that form of cancer, and possibly humans with a similar form of cancer [8,9,17]. A drug which inhibits cox-2 (the nonselective cox inhibitor, piroxicam) is already in use in pet cats [18,19]. Although piroxicam is not FDA approved for use in cats, the drug is being prescribed for cats with a variety of conditions, and is thought to be generally well tolerated [18,19]. The pharmacokinetics of piroxicam in normal cats have been reported [19,20]. Regression of cutaneous SCC in two cats and delay in progression of OSCC in two cats have been noted following piroxicam treatment (unpublished data, Knapp and DiBernardi). This information, collectively, provides justification and lays some of the ground work for a clinical trial of piroxicam in pet cats with OSCC. Before undertaking such a study, however, it was considered appropriate to first determine the extent to which the target for piroxicam treatment (cox-2) is expressed in feline OSCC and to confirm the dosage of piroxicam to be used. This study, therefore, was
conducted to assess: (1) cox-2 expression in feline OSCC, (2) the pharmacokinetics of piroxicam in carcinomabearing cats, and (3) the cox-2 inhibitory activity (measured as in vitro PGE2 release from peripheral blood monocytes) of piroxicam at the various concentrations which were recorded in carcinoma-bearing cats.
2. Materials and methods 2.1. Expression of cox-2 in feline OSCC archival tissues Thirty-four paraffin embedded archival tissue samples of feline OSCC and five samples of normal oral mucosa from non-tumor bearing cats were evaluated for cox-2 protein by immunohistochemistry (IHC). Tissue from a canine prostatic adenocarcinoma served as a positive control. A specific cox-2 antibody (MF243) was generously provided by Drs. Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Doval, Quebec, Canada). MF243 was raised in rabbits against ovine placental cox-2, and its selectivity for cox-2 has previously been characterized [21,22]. Fig. 1 demonstrates that this antibody also cross-reacts with feline cox-2 as it recognized a 72,000– 74,000 molecular weight doublet in a cell protein extract from cultured feline fibroblasts. Immunohistochemical staining was performed using the Vectastain ABC kit, as previously described [23]. Briefly, formalin-fixed tissues were paraffin-embedded, and 3 mm thick sections were prepared and deparaffinized through graded alcohol series. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum for 20 min at room temperature. Primary antibody diluted in PBS was applied (1:7500 dilution), and sections were incubated overnight at 4 1C. Control sections were incubated with PBS or with non-immune rabbit serum. After rinsing in PBS for 10 min, a biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) (1:222 dilution) was applied, and sections were incubated for 45 min at room temperature. Sections were washed in PBS for 10 min, and incubated with the avidin DH-biotinylated horseradish peroxidase H reagents for 45 min at room temperature. After PBS wash for 10 min, the reaction was revealed using diaminobenzidine tetrahydrochloride (DAB) as the peroxidase substrate. Sections were counterstained with Gill’s hematoxylin stain and mounted. The percentage of positively staining tumor or normal epithelial cells and the staining intensity were recorded in multiple fields. The staining intensity was categorized as; 0—no staining, 1—weak or equivocal staining, 2—moderate intensity staining or, 3—marked intensity staining.
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Fig. 1. Cross-reactivity of antibody MF243 for feline cox-2. A solubilized cell extract was prepared from cultured feline fibroblasts (obtained from ATCC) and analyzed by one-dimension SDS-PAGE gel and immunoblotting, as previously described [22]. Markers on the left indicate migration of molecular weight standards.
2.2. Pharmacokinetics of the cox inhibitor, piroxicam, in carcinoma-bearing cats Studies in cats were performed with the approval of the Purdue Animal Care and Use Committee. Eight carcinoma-bearing cats were admitted to PUVTH after acquiring informed owner consent. A jugular catheter was placed in each cat. Piroxicam (PCCA, Houston, TX) was given at a dose of 0.3 mg/kg orally. The powder was weighed, and encapsulated to achieve the desired dose for each individual cat. Blood samples were drawn at 0, 1, 2, 4, 12 and 24 h post piroxicam administration. Blood was collected and placed in a serum separator tube, centrifuged at 5000 rpm, at which time the serum was separated and frozen at 20 1C. Serum analysis was generously performed by Pfizer Global Research & Development Laboratories utilizing high performance liquid chromatography (HPLC) and mass spectroscopy. Following thawing and addition of an internal standard (CP-016,460), serum samples (100 ml) were extracted with 2 ml of ethyl acetate. The organic layers were removed and placed under a stream of nitrogen at 40 1C. The residues were reconstituted with 200 ml of an equal
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mixture of solvent A and B (see below). The samples were agitated, centrifuged and transferred to an autosampler apparatus. Analysis was performed on a SCIEX API 150 HPLC/MS system. The HPLC column effluent was directed into the source via an ion spray interface operated at 2000 eV and the mass spectrometer was operated in the negative ion mode. The HPLC system consisted of a Shimadzu SIL-10AD VP auto-sampler and an HP-1100 de-gasser and gradient pump. Chromatography was carried out on a Phenomenex Luna HPLC column with a binary mixture of solvent A (methanol) and solvent B (10 mM NH4OAc). A flow rate of 0.3 ml/min was used for all analyses. The mobile phase initially consisted of solvent A/solvent B (5/95) for 1 min. It was linearly programmed to achieve a mixture of solvent A/solvent B (95/5) over a 3 min period and operated isocratically for 3 min under these conditions. The mobile phase was then linearly programmed to return to the initial mixture of solvent A/solvent B (5/95) over a 1 min period and operated isocratically for 2 min. This gave a total run time of 10 min. Chromatographic results were recorded using Mass Chrom v. 1.1.1, and were integrated for quantification using MacQuan v. 1.6. The standard curve dynamic range was 10– 4000 ng/ml with a correlation coefficient of 0.985 using a regression analysis weighted 1/x2. These data were analyzed with a non-compartmental, extra-vascular pharmacokinetic model using WinNonlin v. 2.1. 2.3. Cox-2 inhibition in vitro The production of prostaglandin E2 (PGE2) in feline peripheral blood monocytes was determined as a measure of cox-2 activity [24]. Mononuclear cell isolation was performed as described by Guptill with minor modifications [25]. Plastic adherence was used to separate the nonadherent lymphocytes from the adherent monocytes. Monocytes were then treated with piroxicam concentrations of: 1 mM (0.33 mg/ml), 3 mM (1.0 mg/ml), 10 mM (3.33 mg/ml), or 30 mM (10 mg/ml) or piroxicam vehicle (control). Lipopolysaccharide (LPS 10 mg/ml) was added to each well to stimulate cox-2 activity. Following 18 h of incubation at 37 1C, the media was collected and stored at 80 1C until analyzed for PGE2 concentration. PGE2 concentrations were determined utilizing a Prostaglandin E Metabolite EIA kit (Cayman Chemical Company, Ann Arbor, MI) that measures the metabolites of PGE2. The manufacturer’s protocol was followed.
3. Results 3.1. Expression of cox-2 in feline OSCC archival tissues Immunohistochemistry (IHC) to detect cox-2 protein was performed on 34 samples of feline OSCC. Of these
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Fig. 2. Expression of cox-2 in feline normal oral mucosa and oral squamous cell carcinoma. Immunohistochemistry was performed on formalin-fixed sections with antibody MF243 (selective for cox-2). An avidin-biotin-peroxidase complex method with Gill’s hematoxylin counterstain was employed. Weak immunoreactivity (note pale brown staining) was noted in the upper layers of the normal oral mucosa (A). The majority of feline oral squamous cell carcinomas had weak cox-2 immunoreactivity (B), while some tumors demonstrated strong cox-2 immunoreactivity (note intense brown staining in C). Negative control sections (PBS alone) had no immunostaining (D).
Table 1 Serum pharmacokinetic parameters of piroxicam in cats following 0.3 mg/kg p.o. Serum pK parameters
Tmax (h) Cmax (ng/ml) AUCClast (ng h/ml) AUC (ng h/ml) AUC (% ext.) T1/2 (h)
Cat number #1
#2
#3
#4
#5
#6
#7
#8
2.0 1032.5 15217 24820 38.7 16.9
2.0 895.9 12257 15378 20.3 10.3
2.0 579.0 10268 27842 63.1 33.6
2.0 1345.1 16726 22005 24.0 11.4
2.50 905.5 13998 19936 29.8 13.3
1.0 937.8 11318 14165 20.1 9.8
2.0 1105.3 16830 25093 32.9 14.9
1.0 783.7 10376 16626 37.6 16.7
34 OSCC samples, 28 displayed some cox-2 immunoreactivity with intensity varying from weak (staining intensity 1) to strong (staining intensity 3) (Fig. 2B and C). The majority (22/28) of cox-2-positive tumors had weak immunostaining that appeared as diffuse cytoplasmic immunoreactivity in tumor cells (Fig. 2B). Six of the OSCC had strong cox-2 staining intensity in the cytoplasm of neoplastic keratinocytes (Fig. 2C). The percentage of tumor cells with cox-2 immunoreactivity ranged from 15% to 80%. Six OSCC tissues had no cox2 immunoreactivity. Weak cox-2 immunoreactivity was observed in epithelial cells in 4 of 5 samples of normal feline oral mucosa. The immunostaining was mostly localized to the upper layers of the mucosa (Fig. 2A).
Avg.
STD
1.81 948.1 13374 20733 33.3 15.9
0.5 225.9 2701 5031 14.0 7.7
3.2. Pharmacokinetics of piroxicam in carcinoma-bearing cats Pharmacokinetic data are summarized in Table 1. The average maximum plasma concentration of 948 ng/ml (0.948 mg/ml) occurred at approximately 2 h post oral piroxicam dosing for most cats. The average serum halflife of piroxicam was 15.9 h. 3.3. Cox inhibition in vitro Cox-2 activity, measured as monocyte production of PGE2, was studied in blood samples from five cats. The IC50 of piroxicam was less than 1 mM (0.33 mg/ml). Piroxicam at a concentration of 1 mM inhibited cox-2
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activity by 84.8710.7%. Piroxicam at a concentration of 3 mM (1.0 mg/ml) inhibited cox-2 activity by 9075.6%.
4. Discussion and conclusions In preparation for possible trials of cox inhibitor treatment in cats with OSCC, the extent of cox-2 expression was assessed in feline OSCC tissues. Cox-2 is thought to be an important target for cox inhibitor treatment of cancer. Most, although not all, studies implicate cox-2 as the major mechanistic target for the anticancer activity of these drugs in cancer patients [2,26–29]. High intensity immunoreactivity to cox-2 was noted in 18% of the feline OSCC samples studied with positive staining in 15–80% of tumor cells in these samples. Although weak immunoreactivity was noted in several other cases, the importance of this is not known, especially in lieu of the finding of weak immunoreactivity in normal keratinocytes in normal oral mucosa. In a previous report, cox-2 immunoreactivity was noted in 2 of 21 feline OSCC samples and was limited to weak to moderate intensity staining in 10% or less of cancer cells [16]. In a second report, cox-2 immunoreactivity was noted in 37 of 55 feline OSCC samples, but staining was present in o10% of cells and was only of mild to moderate intensity [15]. Although our study demonstrated more cox-2 immunoreactivity than previously reported for feline OSCC, it still appears that cox-2 expression in feline OSCC is lower than that in human OSCC where 475% of OSCC samples express cox-2. The cause for this difference in cox expression between species is not known. This does not mean that pet cats with OSCC should not be used for cox inhibitor studies, rather that cox-2 expression in the cancer cells be used as an inclusion criteria for cats to participate in such studies. Our group has previously reported the frequent cox-2 expression in naturally occurring canine OSCC, and dogs could also be considered as subjects for cox inhibitor studies [8,30]. Some of the attraction to studies in pet cats is that OSCC in the cat is more common, more devastating, and more lethal than that in dogs. OSCC in cats has been linked to tobacco smoke exposure (as in humans) [31]. Because of the grave prognosis for cats with OSCC and short survival (1–4 months), pet owners are eager for their cats to participate in treatment trials of potentially beneficial, relatively nontoxic therapy. Piroxicam was selected for study because this drug was already in use in pet cats with a variety of conditions [18], it has been generally well tolerated, and it has been associated with possible anticancer activity. Also, other cox inhibitors were not available for use in cats when this work began. The anticancer
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activity of piroxicam has not been formally studied in pet cats, but some evidence exists that this drug has anticancer activity in cats. Partial remission (450% reduction in tumor volume) of cutaneous SCC has been observed in two pet cats (unpublished data, Knapp and DiBernardi) receiving piroxicam (0.3 mg/kg daily per os). In two cats with locally advanced OSCC, a delay in cancer progression was noted after institution of piroxicam (0.3 mg/kg daily per os) for pain relief, and survival of the cats was longer than expected (unpublished data, Knapp and DiBernardi). In nine other cats with advanced OSCC receiving piroxicam, however, anticancer activity was not observed. Piroxicam pharmacokinetics were determined to help confirm the choice of piroxicam dose for follow up trials. The dosage of piroxicam (0.3 mg/kg daily per os) was selected because this dose has been used in cats, and this dose appears to be well tolerated. In addition, the pharmacokinetics of piroxicam given at this dose to normal non-cancer bearing cats have been previously reported [19,20]. Results in the normal cat were similar to our findings in carcinoma-bearing cats in this study. Although it was expected that carcinoma-bearing cats would metabolize piroxicam in a similar fashion to that of normal cats, it was appropriate to confirm this prior to larger studies of the anticancer activity. The half-life of piroxicam in the blood of carcinoma-bearing cats was estimated to be 15.9 h. The short duration of sample collection relative to the half-life of the drug resulted in all cats demonstrating an extrapolatedarea under the curve (AUC) greater than 20%. Thus, the serum half-life of 15.9 h in this study is considered an estimate. The average peak piroxicam concentration measured in carcinomabearing cats following a single dose of piroxicam (948 ng/ ml) was found to be sufficient to block cox-2 enzyme activity (the major target of cox inhibitor therapy). While it would be helpful to know the piroxicam concentrations achieved in the cancer tissues and the length of time piroxicam remains in cancer tissues, assays to perform such measurements were not available. The results of this study provide insight into the design of trials of cox inhibitor treatment in cats with OSCC. The pharmacokinetics work suggests that the dose of piroxicam in current use in cats (0.3 mg/kg) would be appropriate for use in a clinical trial. Inclusion criteria for the trial should include overexpression of cox-2 in cancer cells from that cat. Although this may only allow inclusion of approximately 20% of cats with OSCC, with the devastating nature of this cancer in pet cats, a potentially beneficial treatment option for 20% of affected cats would represent a notable improvement compared to current therapy [13,14]. Pet cats may benefit from cox inhibitor treatment trials while important information is being generated to define the potential application of this therapy approach for animals and humans.
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Acknowledgements The authors would like to thank Lisa Holeman, Debra Schlittler, Rose Thomas, Sarah Bradley, and Jane Stewart for their assistance with this work and manuscript preparation.
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