In this study, we obtained critical information regarding the PK and toxicity of MnBuOE in normal dogs as a prelude to a planned clinical trial in canine patients with lymphoma.
1/2 of MnBuOE was defined as 7 h via primary elimination and 20 days via secondary processes. Following a multi-dose PK study, the highest recorded tissue drug levels were in the peripheral lymph nodes (3.98–5.99μM), followed by the kidney and liver (2.58, 1.97 μM, respectively).
The most important result from the single-dose 14-day PK study was that the 99% of drug was eliminated from the plasma after 48 h. This implies that, in a multi-dose treatment protocol, the same initial dose may be given every 2 days without the danger of drug accumulation above the established single-dose MTD. The data obtained from 48 h to 14 days suggest a slow elimination from multiple “deep” compartments (cell cytosol and organelles). Another important result is that C
max (4 μM) obtained in this study with dogs is much higher than the value observed in mice [C
max ~0.3 μM (dose adjusted from 6 mg/kg ) and C
max = 1 μM (1 mg/kg, unpublished data)], rats [C
max = 1 μM (1 mg/kg, unpublished data)], and non-human primates [C
max = 1.6 μM (1 mg/kg )]. This can explain the unexpected serious side effects and the lower than expected MTD established in this study with dogs.
Based on the results from the multiple-dose PK study, it was confirmed that the plasma peak concentration is controlled (no accumulation observed over time, Fig. 4a). This suggests that the acute toxicity signs should not worsen over the course of long-term therapy. Tissue analysis revealed accumulation of the drug within organs. Particularly encouraging for this study is that the highest tissue drug levels were observed in lymph nodes. If plasma peak concentration were the only controlling factor for the acute toxicity, the observed tissue accumulation would be only beneficial for the treatment. However, pulse data as well as laboratory assessments suggest that controlling plasma level is not sufficient and that long-term multiple-dose MTD should be lower than 0.25 mg/kg and/or frequency of dosing extended to once-weekly, depending on the application.
As we prepare to move toward testing the utility of adjuvant MnBuOE in the treatment of naturally occurring canine lymphoma, the finding that the highest drug levels were measured in the lymph nodes is particularly encouraging. The high tissue drug levels in lymph nodes have not been documented previously; this was the first animal study to perform such measurements. Although it is a different cell type, we have reported a threefold higher accumulation of manganese porphryins in the nucleus of macrophages compared to the cytosol . It is likely that MnBuOE is also accumulating in the nucleus of lymphocytes. Lymphocytes are a cell type with a very high nuclear to cytosolic ratio and they are tightly packed with a high cell density within the parenchyma of lymph nodes . The preferential accumulation of MnBuOE in lymph nodes is likely due to both the accumulation of the drug within the nucleus of lymphocytes and the high lymphocyte cell density in the lymph node. Although MnBuOE lymph node accumulation was recorded in normal, healthy dogs in this study, it is important to consider that manganese porphyrin compounds also accumulate in tumor tissue. Recently, we demonstrated that MnTE-2-PyP5+, a manganese porphyrin compound similar to MnBuOE, accumulates preferentially in tumor tissue compared to normal tissue . Evidence that manganese porphyrin compounds accumulate in both primary tumor tissue and lymph nodes strengthens the justification to use MnBuOE in a canine lymphoma trial. For these reasons, patients with lymphoma may benefit substantially from adding adjuvant manganese porphyrin to chemotherapy protocols given its chemosensitization properties against lymphoma cells [11, 12]. The high lymph node drug level is also an important finding when considering other types of cancers which metastasize to lymph nodes; for these cancers, improved treatment outcomes may also arise from adjuvant MnBuOE.
The greatest obstacle in treating both human and canine lymphoma is the development of drug resistance. Treatment with adjuvant MnBuOE may be a way around this drug resistance. The combination of manganese porphryins and certain chemotherapy agents creates an environment of high oxidative stress within tumor cells. This pro-oxidant mechanism of chemosensitization by manganese porphyrins creates a scenario whereby lymphoma cells will be less likely to survive and develop drug resistance [11, 12]. This could lead to lengthened remission duration. Yet another advantage to performing comparative oncology studies in dogs is the ability to initiate a Phase I clinical trial prior to patients receiving and failing standard-of-care treatment protocols due to drug resistance. Given that phase I human lymphoma clinical trials typically are only able to evaluate treatment outcomes for patients who have already failed frontline therapy, reports of improved clinical outcomes for dogs treated upfront with experimental therapies, such as adjuvant MnBuOE, could provide support for moving forward with subsequent human clinical trials.
Although there is more flexibility in performing a comparative oncology trial, it is imperative that the canine patients are treated safely and responsibly; after all, these dogs are companion animals. Therefore, before testing MnBuOE in canine cancer patients, it was necessary to understand the safety and optimal dosing regimen in normal dogs. Prior studies in mice demonstrated pharmacological effects in tumor and normal tissues with 1 mg/kg subcutaneous administration of MnBuOE [5, 6, 22]. This dose is well below the MTD established for mice and non-human primates [21, 23]. Thus, 1 mg/kg was selected as a safe initial dose for MTD and single-dose PK studies in dogs which would also provide accurate measurement of MnBuOE in plasma even 14 days after injection. However, the 1 mg/kg dose of MnBuOE induced an anaphylactic drug reaction and a severe, prolonged tachycardia in the dogs. The acute drug reaction was prevented with premedications (steroids, anti-histamines) and the tachycardia was alleviated by reducing the MnBuOE dose. Neither intravenous fluid therapy nor anti-histamine medication affected the tachycardia, indicating that this toxicity is most likely a primary tachycardia and not secondary to hypotension. Consequently, the MTD was lowered to 0.25 mg/kg. Aside from a mild to moderately increased heart rate 1 h post-injection that increased in severity over time, the dogs had no clinical evidence of toxicity throughout the multi-dose PK study at this dose. This change in heart rate throughout the multi-dose PK study is most likely due to accumulation of MnBuOE in the cardiac tissue. The acute anaphylactic drug reaction and tachycardia post-injection have not been described in other species and may be specific to canines.
Laboratory tests performed throughout the studies identified changes to the organ systems functions of the dogs when treated with MnBuOE. Following treatment of MnBuOE as single doses of 1 and 0.25 mg/kg, as well as prolonged treatment in the first multi-dose study, alkaline urine was documented (n = 1–2/3). A slight-to-mild high anion gap metabolic acidosis developed in all three dogs following the 3-week multi-dose PK study. These results combined are indicative of renal damage. One dog developed a urinary tract infection during the study; however, this was most likely secondary to introduction of bacteria into the bladder during urine collection via cystocentesis. To prevent the anaphylactic drug reaction associated with MnBuOE administration, dogs were treated with prednisone and antihistamine medications throughout the multi-dose PK study. Prednisone can induce changes in liver enzyme activity, and, in fact, mildly increased liver enzyme levels were recorded in the three dogs; however, mild hypocholesterolemia was also found in the three dogs, which may indicate that the changes in the liver are due to damage from the MnBuOE.
Histopathologic evaluation of tissues revealed mild to moderate inflammatory and degenerative changes in the kidney, liver, and lungs. Consistent with the laboratory results, acute, mild to moderate tubular degeneration and necrosis of the kidney was reported (n = 3/3), as well as marked hydropic degeneration of the liver (n = 3/3). Interestingly, mild to moderate sterile bronchointerstitial/interstitial pneumonia was discovered (n = 3/3). While MnBuOE causing these changes in the lungs cannot be ruled out, the characteristics of this pneumonia could also point to an acute injury from inhalation of a noxious cleaning agent from the kennel in which they were housed. Finally, mild necrosupporative/granulomatous inflammation was found in the subcutaneous adipose tissue of the injection site (n = 3/3). These findings have not been reported in other species and may be specific to canines.
Again, because manganese porphyrin compounds accumulate in primary tumors and lymph nodes, it may be possible to reduce the treatment dose and/or frequency of administration of MnBuOE for a canine lymphoma trial while maintaining clinical efficacy in order to reduce or prevent the identified toxicities.