Evaluation of Azathioprine on Lesion Severity and Lymphocyte Blastogenesis in Dogs With Perianal Fistulas
Fourteen dogs with perianal fistulas were entered into a prospective clinical study to investigate the effects of long-term azathioprine on clinical outcome and to determine if the clinical results correlated with lymphocyte blastogenesis tests. Complete remission of perianal fistulas was seen in eight (57%) of 14 dogs; partial remission occurred in one (7%) dog; and no response was detected in five (36%) dogs. The results of lymphocyte blastogenesis assays did not correlate with therapeutic response.
Introduction
Perianal fistula, also known as anal furunculosis, is a chronic, progressive condition characterized by ulcerations, sinus tract formation, and concomitant inflammation of the perianal, anal, and perirectal tissues.1,2 The etiology of the disease has not been elucidated, but an immune-mediated process may be the primary cause.3–6 The disease mainly affects German shepherd dogs, although other breeds have been reported.6,7 Ulceration and inflammation of the perianal region results in pain, tenesmus, dyschezia, constipation, bleeding, fecal incontinence, licking, and secondary anal stricture.1,2,5
Therapeutic options include cyclosporine; topical tacrolimus; azathioprine and metronidazole in combination with surgery; prednisone in combination with a novel antigen diet; and various surgical procedures.1,4,5,8–10 Each of these therapies has specific advantages and disadvantages, such as high success rate but concurrent high cost (e.g., cyclosporine), and low cost but lower success rate (e.g., prednisone and dietary management). The cost of cyclosporine is often prohibitive for many dog owners, although use of some generic formulations has reduced the overall cost of therapy. Additionally, concurrent administration of ketoconazole reduces the required dose of cyclosporine and may decrease the overall cost of therapy by 36% to 71% (compared to cyclosporine alone).11
Azathioprine is used in the therapy of a number of immune-mediated diseases and has the potential benefits of being less expensive than cyclosporine and having fewer side effects and greater efficacy than prednisone.9 The metabolites of azathioprine function as purine analogs, thus serving to disrupt deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis.12 Azathioprine primarily suppresses T-cell-mediated immune responses.12 Although uncommon, the major adverse reaction to azathioprine is severe myelosuppression, which can result in severe neutropenia and thrombocytopenia.12 Other side effects include hepatotoxicity and pancreatitis.12
Predicting the response to azathioprine and determining the reasons for therapeutic failure are not well defined. Measurement of red blood cell thiopurine methyltransferase activity can often predict which people will develop therapy-limiting myelosuppression, but it cannot predict therapeutic success.13–15 Lymphocyte blastogenic responses to various mitogens are reliably depressed after 1 week of azathioprine therapy in dogs, although no studies have related the success of therapeutic response to suppression of lymphocyte blasto-genesis.16 Based on a study by Ogilvie et al., it could be speculated that therapeutic response and suppression of lymphocyte blastogenesis may directly correlate.16
The purpose of this study was to investigate the influence of azathioprine on lesion severity and lymphocyte blastogenesis in dogs with perianal fistulas. The working hypothesis was that lymphocyte blastogenesis should be suppressed in those dogs that responded to therapy and be normal in those dogs that failed to respond.
Materials and Methods
Study Design
Thirteen German shepherd dogs and one border collie diagnosed with perianal fistulas that had not previously received immunosuppressive agents were prospectively included in the study, which ran from December 2000 through September 2001. The initial evaluation of all dogs included a history (duration of perianal fistula and associated clinical signs), complete physical examination, complete blood count (CBC), serum biochemical profile, urinalysis, fecal flotation, serum total thyroxine concentration, serum antinuclear antibody (ANA) assay, and lymphocyte blastogenesis assay. The severity of the perianal fistula was subjectively graded as mild (i.e., a few superficial draining tracts involving <180° of the perianal region, no rectal stricture, no anal sac involvement), moderate (i.e., deeper and more widespread draining tracts involving >180° of perianal region, no rectal stricture or anal sac involvement), or severe (i.e., extensive and widespread with deep sinus tracts, involving almost 360° of perianal region).5,17
Azathioprine therapy was initiated at 2 mg/kg per os (PO) q 24 hours, and that dose was continued until a reduction in the size, number, and degree of inflammation of the perianal fistulas was seen or myelosuppression (defined as a total white blood cell count (WBC) <5000 cells/μL, a neutrophil count <3500 cells/μL, or a platelet count <160,000 cells/μL) was detected. The dose of azathioprine was then reduced to 2 mg/kg PO q 48 hours and continued for 12 weeks, as long as myelosuppression did not develop. At the end of 12 weeks, the dose was reduced to 1 mg/kg PO q 48 hours, with a planned duration of therapy of 12 months. Prednisone was also administered at 2 mg/kg PO q 24 hours for the first 2 weeks, followed by 1 mg/kg PO q 24 hours for another 2 weeks, at which time it was discontinued. All dogs were placed on a limited antigen diet.a,b
All dogs were evaluated at 2-week intervals while receiving the initial azathioprine dose. A thorough history, a physical examination of the perianal fistula, and a CBC were performed. The perianal fistulas were graded at the end of the 16 weeks as complete remission (i.e., no active fistulas), partial remission (i.e., reduction in size or number of the fistulas), or no response (i.e., no change in size or number of fistulas, relapse of fistulas, or dog removed from the study because of toxicity from the azathioprine).
Lymphocyte Isolation and Lymphocyte Blastogenesis
Lymphocyte blastogenesis was performed prior to therapy and at weeks 2, 8, and 16, with additional assays if the animal was available for follow-up. Whole blood (10 mL) was collected by venipuncture and placed into glass tubes containing preservative-free heparin. To concentrate the mononuclear cells (lymphocytes and monocytes), the whole blood was diluted 1:1 with 0.01 molar phosphate-buffered saline (pH 7.4), layered onto a density gradient (Ficoll-Paque 1.077),c and centrifuged for 30 minutes at 1200 revolutions per minute.d The cell interface containing mononuclear cells was collected and washed three times in phosphate-buffered saline to remove the density gradient. Contaminating red blood cells were lysed with ammonium chloride (0.8% NH4Cl), and lymphocytes were counted using a Neubauer hemocytometer.e
Lymphocytes were stimulated in vitro with phytohemagglutinin Af or concanavalin A,g which are T-cell-specific mitogens, or with pokeweed mitogen,h which stimulates both T and B lymphocytes. Lymphocytes (5 × 105 cells/mL in RPMI-1640 mediumi containing 10% heat-inactivated fetal bovine serum,j L-glutamine [0.3 mg/mL], penicillin [10,000 units/mL], streptomycin [10 mg/mL], and gentamicin [12.5 mg/mL]) were placed (200 μL) into flat-bottomed, 96-well plates.k Triplicate wells were stimulated with 20 μL of phytohemagglutinin A or concanavalin A (1 μg/mL) or pokeweed mitogen (1:10 dilution). Lymphocyte cultures were incubated for 72 hours at 37°C with 5% carbon dioxide. Negative control cultures consisted of unstimulated lymphocytes. To measure cell proliferation, 0.2 μCi of [3H] thymidine was added to cells 18 hours prior to harvest. The cells were harvested after 72 hours of incubation, using an automated multiwell cell harvester.l The amounts of tritiated thymidine incorporated into the DNA of proliferating lymphocytes were expressed as the radioactive counts per minute, as determined by liquid scintillation counting.m Proliferation was expressed as a stimulation index, which is the ratio of the counts per minute for mitogen-stimulated cells divided by the counts per minute for nonstimulated cells.
Lymphocyte blastogenesis was also performed concurrently on 34 samples from a normal, healthy dog (German shorthaired pointer), which served as controls. The same dog was used for all control samples.
Statistical Analysis
For each time point, mean and median stimulation indices were calculated for all dogs in the study and were compared to the mean and median stimulation indices prior to therapy. The stimulation index was evaluated for each dog at each sampling period and was compared to the stimulation index prior to therapy. To simplify comparisons, the stimulation index at each time point was divided by the stimulation index prior to therapy to derive the stimulation index ratio. A stimulation index ratio of >1.0 was supportive of increased lymphocyte proliferation, and an index 1.0 indicated inhibition of lymphocyte proliferation. For statistical analysis, the stimulation indices for weeks 0, 2, 8, and 16 (when available) were compared as a group for each mitogen, and the stimulation index ratio for each mitogen was compared independently. Dogs were grouped based on complete remission, partial remission, or no response, and stimulation index ratio values were compared within each group for each time point and between each group for the available time points. A nonparametric, one-way analysis of variance was performed with the Kruskal-Wallis test for these comparisons.k,18 Statistical significance was set at P<0.05. Dose rate and treatment duration were compared with the paired t-test.k,19
Results
Fourteen dogs were entered into the study. Dogs ranged in age from 18 months to 8 years, with a median age of 5 years. Body weights ranged from 16 to 43.6 kg, with a median weight of 36.6 kg. Study dogs included five males (three castrated) and nine females (eight spayed). Clinical signs reported by the owners of the dogs included tenesmus (n=7), licking of the perianal area (n=5), painful defecation (n=5), and bleeding (n=3), with several dogs having more than one clinical sign.
Initial physical examination did not reveal significant abnormalities in 13 of the dogs other than perianal fistulas. One dog had severe otitis externa of the left ear. The results of the initial CBC, serum biochemical profile, and urinalysis were normal in all dogs. One dog had a positive fecal flotation for Trichuris vulpis and Ancylostoma caninum. Serum total thyroxine concentrations were normal in all dogs. The serum ANA assay was positive at 1:80 in two dogs and at 1:160 in one dog. The ANA assay was negative in the remaining 10 dogs.
Perianal fistulas were graded as severe in six dogs, moderate in six dogs, and mild in two dogs. Two dogs had a rectal stricture with fecal impaction at initial evaluation. The duration of the perianal fistulas ranged from 3 weeks to 2 years, with a median duration of 7 weeks. In 12 dogs, the perianal fistulas were present for 3 months. The remaining two dogs had perianal fistulas for 5 months and 2 years, respectively. Thirteen of the 14 dogs completed the 16-week study. The dog that did not complete the study died at week 7 with necrosuppurative enterocolitis. This dog had perianal fistulas present for 2 years, along with severe rectal stricture and fecal impaction.
The dose of azathioprine varied from 1.7 to 2.5 mg/kg (median 2.2 mg). The median dose of azathioprine for dogs (n=8) with complete remission was 2.2 mg/kg. One dog with partial remission was treated with 2.2 mg/kg. The median dose of azathioprine for dogs (n=5) with no response was 1.9 mg/kg. The difference in dosages between the complete remission and no response groups was not statistically significant (P=0.14). The duration of azathioprine therapy at 2 mg/kg per day was 4 weeks for the dogs with complete remission and 2 weeks for the one dog with partial remission. For the nonresponders, this dose was given for 6 weeks in three dogs, 4 weeks in one dog, and 2 weeks in one dog. The difference in treatment duration between the complete remission and no response groups was not statistically significant (P=0.37).
Complete remission occurred in three of six dogs with severe disease, three of six dogs with moderate disease, and two of two dogs with mild disease. Of the five dogs classified as nonresponders, bone marrow suppression was a limiting factor in three. None of these three dogs showed more than a partial response initially, and all relapsed on a lower dose of azathioprine. One of these dogs responded to long-term prednisone and remained in complete remission for 16 months (duration of follow-up). Another dog had good response to gold sodium thiomalate (6 months of therapy) and was in remission 2 years after discontinuation of this medication.
Follow-up beyond 16 weeks was available for all eight dogs with complete remission. Four dogs were followed for 20 to 28 weeks after the end of the study, and they remained in remission. Four dogs were followed for >1 year and remained in remission. Two of these dogs remained continuously on azathioprine at 1 mg/kg PO q 48 hours for 12 and 16 months, respectively. In two dogs, azathioprine was discontinued after 12 months. One remained in remission, and the other was switched to topical 0.1% tacrolimus to reduce the expense of treatment. The dog with partial remission was lost to follow-up after 16 weeks. Side effects were minimal for all but one dog and were primarily related to myelosuppression. Myelosuppression occurred in two dogs that experienced complete remission, but the degree of leukopenia and thrombocytopenia was not severe enough to warrant discontinuation of the drug, and cell counts rebounded to within normal limits after a 50% reduction in the dosage. The dog that died of necrosuppurative colitis developed severe leukopenia (WBC 1600 cells/μL; reference range 6000 to 17,000 cells/μL) and moderate thrombocytopenia (84,000 cells/μL; reference range 200,000 to 500,000 cells/μL) at week 6 and died the following week. No other side effects were noted.
Lymphocyte Blastogenesis
Lymphocyte blastogenesis assays were unexpectedly interrupted as a result of arson damaging the equipment necessary to perform the tests, so only 10 dogs were included in this part of the study. One dog had samples assayed at weeks 2, 8, and 16; eight dogs had at least two samples evaluated at these time intervals. Overall, eight dogs had lymphocyte blastogenesis assays performed at week 2, seven dogs had assays performed at week 8, and three dogs had assays performed at week 16. Some dogs had assays performed at odd times, such as week 4 (n=3), week 10 (n=1), and week 12 (n=3).
The stimulation indices for the three mitogens tested are summarized in Table 1. The stimulation index ratios are shown in Table 2. There was no significant difference in stimulation index ratios at weeks 2, 8, or 16, respectively, among dogs with complete remission, partial remission, or no response for any of the three mitogens (P=0.2127 to 0.5257). There was no significant difference in stimulation index ratios at weeks 2, 8, and 16 for dogs with complete remission for any of the three mitogens (P=0.1130 to 0.3186).
Discussion
In this study, azathioprine therapy induced a reasonable remission rate, with 64% of the dogs having a complete or partial remission. This remission rate was much lower, however, than the rates achieved with cyclosporine alone or combined with ketoconazole (96% to 100%). The complete remission rate for azathioprine (57%) in the current study was also lower than that reported for cyclosporine (66% to 85%).4,11,17,20 By comparison, tacrolimus ointment applied topically has achieved an overall improvement rate of 90% and a complete remission rate of 50%, and high-dose prednisone has produced an overall improvement rate of 67% and a complete remission rate of 33%.5,8 An earlier study evaluated azathioprine and metronidazole therapy in five dogs with perianal fistulas and found a reduction in fistula size over a 4- to 6-week period, but no dogs were disease free during treatment periods that ranged from 5 to 24 weeks.9 The dose of azathioprine (50 mg per day) used in that study was lower than the dose used in the current study.
The lymphocyte blastogenesis assay was not an effective tool in evaluating the response to azathioprine in this group of dogs. Although only two dogs with no therapeutic response had lymphocyte blastogenesis assays performed, both showed reductions in the stimulation index ratio during therapy. In contrast, only four of seven dogs assayed that were in complete remission had reductions in the stimulation index ratio. Of the eight dogs that had a 2-week lymphocyte blastogenesis assay performed, four had no reduction in stimulation index ratio, which suggested that the short-term response to azathioprine is variable. These findings contrasted to the findings of Ogilvie et al., which showed a significant suppression in lymphocyte blastogenesis to all three mitogens after 1 week of azathioprine therapy (2.0 mg/kg per day PO) in all dogs (n=6) tested.16 Although concanavalin A and phytohemagglutinin A are T-cell specific and azathioprine is believed to primarily suppress the T-cell response, there was no consistent suppression of these mitogens (in comparison to pokeweed mitogen) in the present study.16
The stimulation index and stimulation index ratio were used to compare lymphocyte proliferation responses. The study by Ogilvie et al. used an absolute number of counts per minute calculated by subtracting the counts per minute of unstimulated cells from the counts per minute of stimulated cells.16 A number of variables may affect the validity of this absolute number, most of which are involved in sample handling and processing. Stimulation index ratios were also likely influenced by a number of unpredictable variables in sample handling and processing. The ranges of results for the stimulation index from the control dog for the three mitogens varied dramatically (concanavalin A, range 1 to 94; phytohemagglutinin A, range 1 to 82; pokeweed mitogen, range 1 to 73). Killingsworth et al. examined lymphocyte proliferation responses to the same three mitogens in dogs surgically treated for perianal fistula and also reported varied responses.21 Although some dogs showed an increase in the stimulation index after surgery, other dogs showed a decrease or no change.21 Killingsworth et al. concluded that depressed lymphocyte proliferation responses occurred secondary to the perianal fistulas. In the present study, the stimulation index was increased dramatically in three dogs by week 8 and in one dog by week 2. The reason for this increase was unknown. The prednisone that was concurrently administered during the first 4 weeks of the study would have been expected to suppress lymphocyte blastogenesis by weeks 2 and 4; however, no consistent suppression was seen.
Conclusion
Fourteen dogs with perianal fistulas were treated with azathioprine with variable success. Adverse effects associated with azathioprine therapy were related to myelosuppression, but they were mild and responded to a reduction in dosage. Measurement of lymphocyte blastogenesis did not correlate with clinical responses to azathioprine.
Hill’s Prescription Diet z/d; Hill’s Pet Nutrition, Inc., Topeka, KS 66601
Eukanuba Response Formula FP; The Iams Company, Dayton, OH 45414
Ficoll-Paque 1.077; Sigma-Aldrich Co., St. Louis, MO 63103
Beckman TJ6 centrifuge; Beckman Instruments, Inc., Fullerton, CA 92634
Neubauer hemocytometer; AO Scientific, Buffalo, NY 14240
Phytohemagglutinin A; Sigma-Aldrich Co., St. Louis, MO 63103
Concanavalin A; Sigma-Aldrich Co., St. Louis, MO 63103
Pokeweed mitogen; Sigma-Aldrich Co., St. Louis, MO 63103
RPMI-1640 medium; Gibco, Invitrogen Corporation, Carlsbad, CA 92008
10% heat-inactivated fetal bovine serum; HyClone Labs, Logan, UT 84321
96-well plate; Corning Inc., Corning, NY 14831
PHD cell harvester; Brandel Inc., Gaithersburg, MD 20877
LS 9800 Liquid scintillation counter; Beckman Instruments, Inc., Fullerton, CA 92634
Prism 3.0; Graph Pad Software, Inc., San Diego, CA 92130
Acknowledgment
The authors thank Wilma Shuman for her technical support in lymphocyte blastogenesis assays.
