Journal of Veterinary Internal Medicine
  • Open Access

Phase I Clinical Trial and Pharmacokinetics of Intravesical Mitomycin C in Dogs with Localized Transitional Cell Carcinoma of the Urinary Bladder

Authors


  • The clinical work was performed in the Purdue University Veterinary Teaching Hospital, Purdue University, West Lafayette, IN (Abbo, Fourez, Knapp). In vitro drug growth inhibition assays were performed in the Knapp Cancer Biology Laboratory, Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN (Knapp, Stewart). Analytical work was performed by the Clinical Pharmacology Analytical Core Laboratory, a core laboratory of the Indiana University Melvin and Bren Simon Cancer Center supported by the National Cancer Institute grant P30 CA082709 (Jones, Masters).

Corresponding author: Dr Deborah W. Knapp, Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026; e-mail: knappd@purdue.edu.

Abstract

Background: Transitional cell carcinoma (TCC) is the most common cancer of the urinary tract in dogs. The most frequent cause of death is urinary obstruction from the primary tumor. Standard medical therapy for TCC is only partially effective.

Hypothesis/Objectives: Intravesical administration of mitomycin C (MMC) in dogs with invasive TCC will result in antitumor activity against the primary tumor and minimal systemic drug absorption.

Animals: Thirteen privately owned dogs with naturally occurring, histopathologically diagnosed TCC of the urinary bladder.

Methods: A prospective phase I trial was performed. MMC was given intravesically (600 μg/mL initial concentration) for 1 h/d for 2 consecutive days each month. The MMC concentration was escalated to a maximum of 800 μg/mL in groups of 3 dogs until the maximum tolerated dose (MTD) was determined. Serum assays for MMC were performed to determine the extent of systemic absorption of the MMC.

Results: The MTD of MMC based on local toxicoses was 700 μg/mL (1-h dwell time, 2 consecutive days). In addition, 2 dogs had severe myelosuppression and appeared to have systemic absorption of MMC. Five dogs had partial remission, and 7 dogs had stable disease.

Conclusions: Intravesical MMC has antitumor activity in dogs with invasive TCC. Further study is needed to determine the cause of the myelosuppression associated with MMC administration, and to develop strategies to minimize this risk.

Abbreviations:
COX

cyclooxygenase

HPLC

high performance liquid chromatography

MMC

mitomycin C

MTD

maximum tolerated dose

TCC

transitional cell carcinoma

Transitional cell carcinoma (TCC) is the most common cancer of the lower urinary tract in dogs.1,2 Distant metastasis is reported to occur in up to 50% of affected dogs. In the majority of dogs, however, the most common cause of death is progression of local disease leading to urinary tract obstruction.1 Standard medical therapy for TCC is only partially effective, and overall response rates can be disappointing.1–9 The unique properties of the urinary bladder, including access via catheter and a “closed” organ with minimal absorption of agents “through” the bladder wall, make intravesical therapy a promising treatment approach for dogs with TCC confined to the bladder.10 Intravesical therapy should allow for increased drug concentrations to be delivered to the site of the tumor while minimizing the risk of systemic absorption and subsequent adverse effects. One of the agents studied in intravesical therapy in humans is the alkylating agent mitomycin C (MMC).11–15

Intravesical MMC has been evaluated in humans with low grade superficial TCC and high-grade carcinoma in situ.16–19 Complete response rates in humans have exceeded 50% with no patients having any reported severe systemic toxicosis. Intravesical MMC (up to 1 mg/mL, dwell times 5–120 minutes) has been administered to healthy laboratory dogs with no biochemical or hematological changes observed.10,20 The lack of systemic toxicoses is encouraging because this not only suggests a good safety profile for single agent use, but also indicates that MMC could be evaluated in the future in combination with other systemic therapies aimed at treating metastatic disease.

Although intravesical therapy is widely used to treat superficial bladder cancer in humans, it has not been evaluated in people or dogs with the high-grade muscle invasive form of bladder cancer. The goals of this prospective phase I clinical trial in dogs with TCC were to determine (1) the maximum tolerated dose (MTD) of intravesical MMC, (2) the serum pharmacokinetics of MMC after intravesical administration, and (3) the anticancer activity of intravesical MMC.

Materials and Methods

In Vitro Growth Inhibition Assays to Select the Initial Concentration of MMC for the Clinical Trial

The selection of the starting MMC intravesical concentration was based on results from laboratory dog studies,10,20 and from in vitro growth inhibition assays. Three types of assays were performed: (1) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which detects mitochondrial activity in living cells, (2) sulforhodamine B assay, which allows quantification of cell protein and extrapolation of cell number, and (3) a clonogenic assay. For the MTT assay, canine TCC cells (K9TCC, K9TCC-PU-Ab, and K9TCC-PU-AxA)21 were plated in 96-well plates at a concentration of 3 × 103 cells/well in 50 μL media,a 10% fetal bovine serum,b 2 mM l-glutamine.c Cells were allowed to grow (37°C, 5% CO2) for 24 hours before treatment. Serial dilutions of MMCd in media (50 μL/well) were added to the wells. After 1 hour, the treated media was removed, and the wells were washed with PBS. New media (100 μL) was added, and the plates were returned to the incubator for 48 hours. After incubation, the plates were examined microscopically and assessed for surviving cells by adding 20 μL of MTT, 5 mg/mL in watere to each well for 1 hour. Media was removed, and the resulting dye formed was dissolved with 100 μL DMSOf per well. The plates were read on a spectrometer at 570 nm minus a 650 nm correction. The percent survival was calculated by dividing the corrected optical density (OD) reading corresponding to a specific concentration of MMC with the OD of the untreated control.

The sulforhodamine B assay procedure was adapted from the NCI-60 DTP Human Tumor Cell Line Screen (http://dtp.nci.nih.gov/branches/btb/ivclsp.html). Canine bladder cancer cells (K9TCC, K9TCC-PU-In, K9TCC-PU-Sh, and K9TCC-PU-AxA)21 were plated at 3,000–5,000 cells per well in 96-well plates with DMEM/F12 medium containing 5% fetal bovine serum and 2 mM l-glutamine, and incubated at 37°C, 5% CO2 for 24 hours before the addition of various concentrations of MMC. MMC was left in the wells for 1 hour, after which media and drug were removed, wells were washed with PBS, and 100 μL of fresh media placed in wells. Specific wells were fixed at this 24-hour time point for the calculation of time zero (Tz). The plates were incubated for another 48 hours, and cells in remaining wells were fixed by the addition of TCA. The plates were washed, and cells were stained with sulforhodamine B solution. After washing the unbound dye away, the stain was solubilized with 200 μL Tris buffer, and the plates read at 490 nm. The readings were averaged for each dilution of the drug to calculate Ti. The control wells with no drug treatment were averaged to calculate C. Percentage growth inhibition was calculated as follows: [(TiTz)/(CTz)] × 100 for concentrations for which TiTz, and [(TiTz)/Tz] × 100 for concentrations for which Ti < Tz.

For the clonogenic assay, the K9TCC cell line was selected because of its colony forming growth characteristics suitable for clonogenic assay. Briefly, 500 single cells per well were seeded into a 6-well plate with DMEM/F12 media containing 10% fetal bovine serum and 2 mM glutamine. Plates were incubated at 37°C at 5% CO2. After 24 hours MMC (6 concentrations ranging from 0 to 3.3 μg/mL) in media was placed in the wells and left for 1 hour at 37°C. The MMC was then removed, the plates were washed with PBS, and fresh media was added. The plates were incubated under the initial conditions while being observed for colony formation. After 10 days, the media was removed, and 1 mL of phosphate buffered formalin (3.7% formaldehyde in 0.1 M phosphate buffer) with 2% crystal violet was added to each well. The plates were incubated at room temperature for 30 minutes and then gently rinsed with water. Colonies of >50 cells/colony were counted in each well.

Clinical Trial

A prospective, phase I clinical trial was performed at Purdue University with the approval of the Purdue Animal Care and Use Committee.

Animals and entry requirements. Privately owned dogs with naturally occurring, histopathologically confirmed TCC, localized measurable tumor in the lower urinary tract (urinary bladder, prostate, urethra), expected survival of at least 6 weeks, and informed pet owner consent in writing were included in this study. Prior treatment with other drugs such as chemotherapy or cyclooxygenase (COX) inhibitors did not exclude dogs from participation. Dogs that were receiving COX inhibitors before the trial were allowed to remain on COX inhibitors if their bladder cancer was progressing on the COX inhibitor treatment and if they benefited from the drug for pain relief from comorbid conditions such as arthritis. Dogs with prostate and urethral extension of the bladder cancer were also enrolled in this study. This was allowed because tumor regression had been observed at these sites after intravesical treatment in preliminary work.

Animal evaluation and monitoring. Major evaluations including tumor staging were performed before treatment and at 8-week intervals during treatment at the Purdue University Veterinary Teaching Hospital (PUVTH). Major evaluations consisted of physical examination including rectal exam; radiography of the thorax (right and left lateral, and ventrodorsal views) and abdomen (right lateral and ventrodorsal views); abdominal ultrasonography (to look for evidence of enlarged lymph nodes and masses indicative of metastases); and bladder imaging. Bladder imaging primarily consisted of cystosonography;3 however, in some cases additional imaging tests were utilized, such as computed tomography or double contrast cystogram.3 The estimated tumor volume was recorded. The same imaging modality was used in the same patient for each visit. Minor evaluations (physical examination, cystosonography, CBC, platelet count, and serum biochemistry profile) were performed at the PUVTH at approximately 2-week intervals (owner schedule allowing) while undergoing therapy.

Treatment. MMCg was prepared for intravesical therapy as summarized in Table 1 based on MMC concentration to be achieved in the bladder and the size of the dog. The starting dose of MMC (concentration of drug in the intravesical solution) was to be one associated with acceptable toxicoses in laboratory dogs,10,20 and one that inhibited growth of the canine TCC cells in culture, ie, one inhibiting growth by approximately 100% in cells in culture. The initial study design called for 3 dogs to be treated with the starting dose (1-h dwell time; 2 consecutive days per month) delivered through a urinary catheter. After the 60-minute dwell time, the MMC was removed via urinary catheter. The treatment cycle (2 consecutive days of treatment in a month) was repeated at 4-week intervals. Note, the schedule of administering intravesical therapy 2 days per month had been well tolerated with another investigational drug given by intravesical route to dogs with TCC (D.W. Knapp and A.H. Abbo, unpublished data) and was one considered feasible for frequency of hospital visits by most pet owners. The plan was to increase the MMC concentration by 100 μg/mL for each group of 3 dogs. If, however, the initial concentration was associated with unacceptable toxicoses, then the concentration would be reduced by half of the starting concentration for 3 dogs, and then increased by 100 μg/mL in each subsequent group of 3 dogs until the MTD was determined. If a dog had received 3 treatment cycles of MMC without evidence of toxicoses and without tumor regression, then the dose of MMC was escalated in that dog; thus dose escalation occurred within as well as between dogs in treatment groups. Pet owners were requested to not give other cancer “treatments,” including herbal supplements and other “holistic” medications.

Table 1.   Preparation of mitomycin C (MMC) for intravesical administration.
Body
Weight (kg)
Total Volume
Saline/MMC (mL)
Mg MMC
(mg)
300 μg/mL
 ≤8257.5
 9–405015
 >407522.5
400 μg/mL
 ≤82510
 9–405020
 >407530
500 μg/mL
 ≤82512.5
 9–405025
 >407537.5
600 μg/mL
 ≤82515
 9–405030
 >407545
700 μg/mL
 ≤82517.5
 9–405035
 >407552.5
800 μg/mL
 ≤82520
 9–405040
 >407560
1000 μg/mL
 ≤82525
 9–405050
 >407575

Toxicoses and defining the MTD. Toxicoses of MMC were expected to be possible through at least 2 mechanisms: (1) direct irritation to the bladder and (2) systemic absorption leading to myelosuppression and organ dysfunction. In order to monitor for toxicoses, dogs were evaluated with a complete physical examination the day of treatment, and vital signs (temperature, pulse, and respiratory rate) were recorded 2 or more times during the days of treatment and while at the PUVTH. Owners were asked to keep a log of abnormalities that were noted while the dogs were at home. A CBC, platelet count, and serum biochemistry panel were performed immediately before MMC administration, and a CBC and platelet count were performed 7–10 days after treatment.

Criteria established by the Veterinary Cooperative Oncology Group were used to categorize bone marrow, renal, and gastrointestinal (GI) toxicoses.22 Criteria were established for the study to define urologic toxicoses (Table 2). The following adverse events were considered unacceptable toxicities: (1) grade 4 renal, GI, hematologic, or urologic toxicoses in any dog in a dose group of 3 dogs, or (2) grade 3 renal, GI, hematologic, or urologic toxicoses in at least 2 of 3 dogs in a dose group of 3 dogs. If unacceptable toxicoses were noted, then the next lowest dose (concentration) was given to a total of 6 or more dogs. The MTD was reached if 0 of 6 dogs had grade 4 toxicoses, and if no more than 1 of 6 dogs had grade 3 renal, GI, bone marrow, or urologic toxicoses.

Table 2.   Criteria to categorize urologic toxicosis.
 Grade 1Grade 2Grade 3Grade 4
Character of urinationNo strainingMinimal straining, attempts to urinate 1–3 times at time of urination, slightly reduced stream, worsened signs persist ≤2 daysModerate straining, attempts to urinate >3 times at time of urination, moderate reduction in stream, worsened signs persist >2 daysExcessive straining, makes repeated attempts to urinate, severely reduced urine stream, worsened signs persist for >2 days
Frequency of urination
 DayDog asks to be let out more frequently following treatment, but <6 × per day; any change in frequency last <2 daysDog asks to be let out 6–10 × per day, any change in frequency persists for ≤2 daysDog asks to be let out 11–15 × per day, change in signs persists for >2 daysDog asks to be let out >15 × per day, change in frequency persists for >2 days
 NightHolds urine at nightHolds urine at nightNot able to hold urine overnightNot able to hold urine overnight
Blood in urineNoneFew drops or slight red tinge; change in signs persists ≤2 daysModerately discolored with blood, change persists >2 daysBlood clots, urine markedly discolored/darkened for >2 days

Tumor responses. Tumor responses were defined as follows. Complete remission was defined as the complete resolution of all clinical, radiographic, or ultrasonographic evidence of tumor. Partial remission (PR) was defined as ≥50% reduction in estimated tumor volume and no new tumor lesions. Stable disease (SD) was defined as <50% change in tumor volume and no new tumor lesions for a period of at least 4 weeks. Progressive disease (PD) was defined as ≥50% increase in estimated tumor volume or the development of new tumor lesions. The progression-free interval was defined as the time from the 1st day of treatment until PD was noted. Survival time was defined as the time from the 1st day of MMC treatment until the dog's death.

Pharmacokinetic Analyses

Blood samples for measurement of serum MMC concentrations were collected at 0, 15, 30, 45, 60, 90, 120, 180, and 240 minutes, and at 24 hours after MMC administration. The serum was collected, and samples were stored at −80°C until analysis. MMC was purchased from A.G. Scientific,h and clarithromycin was purchased from Sigma.i MMC was quantified by high performance liquid chromatography (HPLC)-MS/MSj with clarithromycin as an internal standard. Serum (100 μL), with supplemented clarithromycin, was precipitated with acetone and then centrifuged at high speed. The upper phase was transferred to a separate tube and evaporated to dryness. The tube was reconstituted with mobile phase and an aliquot was injected into the HPLC-MS/MS. MMC and clarithromycin were separated by reverse phase HPLC with acetonitrile: ammonium acetate as the mobile phase. The Q1/Q3 for MMC and clarithromycin were 335/242 and 749/158, respectively. The lower limit of quantification for this assay is 100 pg/mL. Because of the nature of drug delivery and the known variability in the length of time for drug absorption and degree of drug absorption, classical pharmacokinetic analyses would not apply. The data were analyzed in 3 ways: (1) peak concentration of MMC in the blood stream, (2) comparison between the known MMC concentration in the bladder and the peak MMC concentration measured in the blood stream, and (3) the length of time that MMC remained detectable in the blood stream.

Results

In Vitro Growth Inhibition Assay Results and Selection of Starting MMC Dose

In the MTT assay, the IC50 ranged from 3.8 to 62.5 μg/mL MMC across cell lines, and complete inhibition occurred at 250–500 μg/mL MMC. In the sulforhodamine assay, the IC50 ranged from 0.3 to 1.8 μg/mL MMC, with complete inhibition observed at 2.7–3.2 μg/mL MMC (Fig 1). In the clonogenic assay, the IC50 was 0.2 μg/mL. Absence of any colonies was observed at 1.7 μg/mL. Based on the in vitro assay data as well as previously published laboratory dog data, the starting concentration of MMC was 600 μg/mL.

Figure 1.

 Inhibition of proliferation of canine transitional cell carcinoma (TCC) cells in vitro determined by sulforhodamine B assay. The IC50 (determined from the growth inhibition curves) ranged from 0.3 to 1.8 μg/mL for the 4 canine TCC cell lines evaluated.

Clinical Trial

Subject characteristics. Thirteen dogs with TCC were treated with intravesical MMC. Breeds were Scottish Terrier (n = 5), Beagle (n = 3), West Highland White Terrier (n = 1), Shetland Sheepdog (n = 2), mixed breed (n = 1), and Rhodesian Ridgeback (n = 1). Body weight of the dogs ranged from 10.2 to 50.9 kg (median 13.5 kg, mean 16.3 kg). There were 9 neutered male and 4 spayed female dogs ranging in age from 5 to 14 years (median 9 years). Six dogs had received prior chemotherapy including doxorubicin, carboplatin, cisplatin, gemcitibine, or mitoxantrone; and 9 dogs had received prior COX inhibitor therapy. Two dogs who had received prior COX inhibitor treatment and whose tumors were progressing on the COX inhibitors continued to receive a COX inhibitor while receiving MMC. For these 2 dogs, the pet owners requested continued COX inhibitor treatment to help relieve pain associated with the cancer and concurrent arthritis.

Defining the MTD of Intravesical MMC Therapy. Intravesical MMC therapy was administered in 50, 2-day cycles to a total of 13 dogs (Table 3). The 1st dog to receive 600 μg/mL MMC (60-min dwell time, 2 consecutive days) experienced severe unacceptable (grade 4) GI and bone marrow toxicoses. Anorexia was detected 3 days after beginning MMC, followed by vomiting and diarrhea that worsened daily. The dog was hospitalized 7 days after beginning MMC for fluids administered IV and supportive care. The neutrophil count at that time was 120/mm3. The dog fully recovered over the next 3–5 days. The owner reported that the dog had received multiple “holistic supplements” and “home remedies,” and it was not known if these contributed to the adverse events. After the trial design, however, the MMC concentration was reduced to 300 μg/mL in the next 3 dogs treated.

Table 3.   Number of dogs receiving mitomycin C (MMC), and number of treatment cycles delivered.
MMC Concentration
(μg/mL)
Number of DogsNumber
of Cycles
30038
40024
50038
60047
700617
80036

Three dogs received 300 μg/mL MMC, and this dose was given for 8 treatment cycles. Signs of intoxication were mild to none and consisted of mild transient increase in hematuria and stranguria (grade 1–2), which resolved within 24–48 hours. Dose escalation was continued, increasing the MMC concentration by 100 μg/mL in each new dose group. Even at 500 μg/mL of MMC, toxicoses remained mild to none and consisted of mild transient increase in hematuria and stranguria that resolved in 24–48 hours after treatment. Serial CBCs indicated no change in neutrophil or platelet counts. Because it was not known if the initial dog treated had become ill from the MMC or from other products given by the dog's owner, the decision was made to continue the MMC dose escalation. Three newly enrolled dogs were treated with 600 μg/mL MMC. These dogs received 6 cycles of treatment, with only minimal signs of toxicosis (transient increase in hematuria and stranguria). Continuing with dose escalation, 3 dogs received 700 μg/mL MMC, and no toxicosis other than transient bladder irritation occurred. The dose was further escalated to 800 μg/mL MMC, and 3 dogs were treated. Two of the 3 dogs receiving 800 μg/mL MMC had marked bladder discomfort within 15 minutes of drug instillation into the bladder indicating marked local irritation. This was considered unacceptable toxicosis and beyond the MTD. No other toxicoses such as myelosuppression or GI toxicosis were detected in the dogs receiving 800 μg/mL MMC. To determine the MTD, additional dogs were then treated at 700 μg/mL MMC. A total of 6 dogs received 700 μg/mL MMC, and 17 cycles were administered at this dose. Based on acceptable signs of bladder toxicoses, 700 μg/mL was defined as the MTD.

As treatments were continued, however, a 2nd dog developed grade 4 myelosuppression. This occurred after the 4th cycle of MMC (700 μg/mL). The neutrophil count was 180/mm3 on day 7, and was normal on day 14. The platelet count was reported as adequate. No clinical signs of toxicoses were noted.

Two other possible adverse events were noted. Two dogs developed erythematous or bruised appearing lesions on the end of the prepuce (1 dog treated with 500 μg/mL MMC) or penis (1 treated with 700 μg/mL MMC). It was thought that this was possibly due to irritation from MMC spilling into the prepuce at the time of treatment.

Another adverse event that was noted in 1 dog was uroabdomen that was observed 2 days after MMC treatment. The bladder had filled normally during MMC treatment. It was not known if the uroabdomen was caused by tumor-related bladder damage, catheter injury, or other causes. The dog's owner requested euthanasia of the dog and did not elect for further medical care or for necropsy after euthanasia.

Tumor Response to Intravesical MMC in Dogs with TCC. The tumor response to MMC was determined in 12 dogs and included PR in 5 dogs and SD in 7 dogs. The 1st dog treated did not live 4 weeks to allow the scheduled follow-up exam. This dog, which was the first to have severe toxicoses, was euthanized 21 days after beginning MMC. An ultrasound exam performed before death showed no change in tumor size. The pet owner requested euthanasia because of the dog's continued clinical signs and the owner's unwillingness to take risk with any further therapy. Of the 5 dogs that had PR, the MMC doses included 300 μg/mL (1 dog), 600 μg/mL (1 dog), and 700 μg/mL (3 dogs). The dog that had remission with 300 μg/mL MMC had failed mitoxantrone and COX inhibitor treatment before enrolling in the MMC trial. Similarly, 4 of the dogs that had SD with MMC treatment had failed extensive other therapies including cisplatin, carboplatin, gemcitabine, mitoxantrone, doxorubicin, and COX inhibitors before enrolling in the MMC trial. The median progression-free interval was 119.9 days (mean 109 days, range 7–204 days) from the initiation of MMC therapy. The median survival time from the start of MCC therapy until death was 223 days (mean 239 days, range 21–482 days) with 20% alive at 1-year postinitiation of MMC treatment. Four dogs received other therapy after failing MMC, including an investigational metronomic chemotherapy protocol, COX inhibitors, and investigational 5 azacitidine.

Pharmacokinetics of Intravesical MMC in Dogs with TCC

Pharmacokinetic analyses were performed in 6 dogs during 8 cycles of treatment (2 dogs provided samples in 2 different cycles). The blood samples were collected after the 1st dose of MMC in the 2-dose cycle, and reflect the absorption after a single dose of the drug. Blood samples were collected after 300, 400, 500, 600, and 700 μg/mL MMC. The results are summarized in Figure 2. No correlation between dose administered and systemic absorption of MMC was noted in any dog. Peak serum MMC concentrations ranged from 2.3 to 41.9 ng/mL and were not correlated to dosage. Serum concentrations of MMC were calculated to be 16–174 × 103 times lower than in urine. MMC was detected in serum for up to 24 hours after administration (range 3–24 hours).

Figure 2.

 Serum concentrations (ng/mL) of mitomycin C (MMC) measured by high-performance liquid chromatography (HPLC)-MS/MS from dogs receiving intravesical MMC (1-hour dwell time). Samples were collected from 8 treatment cycles in 6 dogs.

Discussion

The results of the study were encouraging in that of 13 dogs enrolled, 5 had PR and 7 had SD. Most of the dogs had failed previous therapy. The tumor response after MMC compared favorably with results from other treatments.1 In the 6 dogs in which blood concentrations of MMC were measured, the concentration of MMC in the bladder was up to 174 × 103 (range 16–174 × 103) times greater than that in the blood. Minimal signs of toxicoses were observed in most dogs at MMC concentrations below 800 μg/mL. In addition, it is important to acknowledge that this is the 1st step in testing intravesical therapy in dogs with TCC. It is likely that the therapy could be optimized for greater efficacy such as by increasing dwell time or number of treatment days per month.

The one main concern in the study results was the apparent systemic absorption of MMC in 2 dogs. The 1st dog treated developed severe GI upset and myelosuppression. At that time, it was not known if this was due to the MMC or the “holistic” treatments the dog received from its owner, or a combination of the two. Once myelosuppression was observed in a 2nd dog, however, it was considered more likely that systemic drug absorption had occurred in both dogs. The apparent systemic absorption was not related to dose or cycle. In the 1st dog, the apparent drug absorption occurred after the 1st cycle of treatment. In the other dog, the severe myelosuppression was not noted until after the 4th cycle of treatment. The dogs that had blood samples analyzed for MMC concentrations had minimal absorption of the drug into the blood stream, but the dogs who developed myelosuppression did not happen to be ones who had blood samples drawn for pharmacokinetics. Therefore, although it was considered likely, it could not be proven that systemic MMC absorption was the cause of the myelosuppression in these dogs.

Several factors could theoretically contribute to systemic drug absorption from intravesical administration, including tumor size and vascularity, increased vascularity due to infection, and possibly penetration of a blood vessel by the urinary catheter. There were not any obvious differences in tumor size or other measurable factors between the 2 dogs who had myelosuppression and those who did not have myelosuppression after MMC. Currently it is not possible to know if a given dog is at high risk or not for drug absorption after intravesical therapy before administering the drug.

Although the dogs that developed myelosuppression responded well to supportive care, the possibility of more severe myelosuppression, that could be lethal in some dogs, presents a serious issue regarding intravesical MMC therapy in dogs with invasive TCC. For this reason, intravesical MMC might not be the best option for routine frontline use in dogs with TCC. In cases where other therapies have failed, the clinician and pet owner should consider the potential risk and benefit of intravesical MMC when making decisions regarding further treatment. Further study is needed to define the cause(s) of the myelosuppression, and to determine ways to minimize the risk of intravesical therapy. With the risk of unpredictable systemic drug absorption, intravesical therapy could evolve in 2 directions. First, protocols could be developed that include doses of “toxic” compounds (such as MMC) that would be tolerated in the unlikely event that most or all of the drug was absorbed into the blood stream. For example, based on the in vitro growth inhibition assay data of MMC and what is known about the toxicoses of IV doses, it could be possible to give MMC at a dose in the bladder that should have some antitumor effects without posing the risk of lethal toxicoses should it be absorbed into the blood. The peak blood concentrations measured in dogs in this study ranged from approximately 2 to 42 ng/mL. For comparison, the systemic concentration of MMC known to cause myelosuppression in humans is 400 ng/mL.18 When designing intravesical therapy, however, consideration must be given to the fact that MMC does not penetrate very effectively into bladder tissues.10 In 1 study, MMC concentrations dropped by 50% for each 0.5 mm increase in depth into the tissues.10 Therefore, high MMC concentration at the surface of the tumor is thought to be essential for even modest drug levels being achieved deeper into tissues.

A 2nd way that intravesical therapy protocols could evolve would be to utilize “nontoxic” drugs, ie, drugs that are not toxic even when given systemically. Although nontoxic drugs could be given systemically, they would also be candidates for intravesical administration. The advantages of delivering these drugs by intravesical route would be to achieve maximum drug concentration right at the tumor where it is needed the most, and to possibly reduce costs by using less drug than if it were to be delivered systemically.

The study results revealed 2 other types of adverse events that can occur with intravesical therapy. The finding of bruises or erythematous lesions on the penis and prepuce (1 dog each) suggest the possibility that the MMC could be irritating if allowed to pool in the prepuce. Thus, care should be taken to limit contact of the penis and prepuce with drugs such as MMC. In a different dog, uroabdomen was noted 2 days after intravesical therapy. It was not known if this was a consequence of tumor growth affecting the bladder wall, dying tumor tissue leaving a weakened area of bladder wall, injury from catheterization, or other causes. This finding still serves as a reminder of the importance of careful catheterization technique in dogs with TCC.

This study was a 1st step in the evaluation of intravesical therapy in dogs with TCC. In addition to generating information for use in veterinary oncology, this study provides information to be considered as the use of intravesical therapies is expanded in humans. To date, intravesical therapy in humans has been directed almost entirely at superficial TCC. Cystectomy has been the front line treatment for invasive TCC. As options to avoid cystectomy are explored, however, intravesical therapy could gain more attention. The results of this study suggest that caution be taken for possible systemic drug absorption in invasive TCC, regardless of the species.

In conclusion, the study results of intravesical MMC treatment were promising in regards to antitumor activity and low toxicoses in most dogs. Further study is needed to determine the cause of the severe myelosuppression associated with MMC administration observed in 2 dogs in this study, and to develop strategies to minimize this risk.

Footnotes

aDMEM/F12, Cellgrow, Manassas, VA

bHyclone, Logan, UT

cCellgrow

dSigma, St Louis, MO

eThiazolyl blue tetrazolium bromide, Sigma

fDimethyl sulfoxide, Mallinckrodt, Phillipsburg, NJ

gMutamycin, Bristol-Myers Squibb Co, Princeton, NJ

hA.G. Scientific, San Diego, CA

iSigma

jAPI 4000, Applied Biosystems, Carlsbad, CA

Acknowledgments

This study was supported by a grant from the American Kennel Club, Canine Health Foundation, and by funds from the Department of Veterinary Clinical Sciences, Purdue University.

Ancillary