Pharmacokinetic and safety in normal dogs
The pharmacokinetic parameters achieved in dogs were comparable to those in humans (Table
6). This justifies the dose of 150 mg/kg used in dogs in this study. Because higher levels of taurolidine were achieved in dogs, the dose in dogs could be decreased if the goal is to achieve same levels as in humans. There is also a 300 mg/kg dose that is reported in humans [
8,
17] which in all likelihood leads to higher concentrations of taurolidine than is reported in the pharmacokinetic studies that used a dose of about 75 mg/kg [
20,
21]. Although the human values are only estimations based on a 66 kg body mass, several differences between dog and human are noteworthy. Dogs appear to have a smaller Vss than humans with longer t1/2, CL and AUC which provides some explanation for the higher levels of taurolidine achieved in dogs.
Table 6
Comparison of pharmacokinetic parameters in dogs and humans after taurolidine infusion
Cmax (μg/mL) | 140.5 ± 40.3 | 95.8 ± 21.8 |
t1/2 (hr) | 3.4 ± 0.4 | 1.7 ± 0.1 |
AUC (μg h/mL) | 455 ± 200 | 249.2 ± 58.4 |
CL (L/hr/kg) | 0.40 ± 0.20 | 1.28* |
Vss (L/kg) | 1.39 ± 0.85 | 3.18* |
Healthy dog#1 achieved a considerably lower Cmax and Area Under the Curve than all other dogs. It is unknown why this was the case. Pharmacokinetic studies done on a larger number of dogs are necessary to try to answer this question.
Healthy dog #1 also manifested an allergic-like reaction within a minute of starting the infusion of taurolidine containing PVP. The reaction was attributed to the PVP. The dog received a total dose of 6.5 mg/kg of PVP. PVP injected in dogs causes immediate release of histamine systemically and at high dose (100 mg/kg) prolonged hypotension [
25‐
27]. In the dose range of PVP that the dog in our study received, the response in dogs is variable but can include labored respiration, unsteady gate, shaking and scratching of the head [
27]. The clinical signs manifested in the dog of this study were partly similar in nature (agitation, pawing at and shaking its head, looking disoriented). This same dog, and all other dogs in this study, did not have this type of reaction or any other signs of an allergic reaction when given the PVP-free solution.
Because of the allergic reaction to PVP in dogs, we had to administer a PVP-free solution of taurolidine. PVP increases the stability of taurolidine in aqueous solution [
28]. In aqueous solution taurolidine breaks down into and becomes in equilibrium with taurultame and taurinamide which eventually leads to the release of formaldehyde. PVP helps to push the equilibrium towards taurolidine such that in the presence of PVP, there is less formaldehyde in solution [
28]. The therapeutic implications of a PVP-free solution of taurolidine are unknown. It is not determined which molecule exerts the therapeutic benefit of taurolidine between taurolidine itself, taurultame, taurinamide or formaldehyde. In our
in vitro experiments, PVP-free taurolidine was effective at killing OSA cells below the serum concentrations achieved in the healthy dogs [
19].
Healthy dog #4 had neutropenia following the infusion of taurolidine. Whether the taurolidine played a role in the presence of neutropenia is difficult to ascertain. Seven months before the infusion, the dog was mildly neutropenic when comparing to the normal range in our laboratory. Also the neutropenia observed after the infusion of taurolidine was very mild (grade 1) so it may be that this dog was normally waxing and waning around the low end of the normal range, mostly running at a lower neutrophil count than the normal range. A bone marrow aspirate determined that the lower neutrophil count was not due to bone marrow suppression. In a study in rats, the administration of taurolidine did not affect leucopoiesis [
29]. The dog was monitored daily for over 3.5 years after the administration of taurolidine and remained healthy. It is unlikely that taurolidine had an effect on the neutrophil count of this dog.
There is no consensus on what blood pressure value constitutes hypertension in dogs [
30,
31]. It has been suggested that hypertension occurs when systolic blood pressure is ≥ 150 to 180 mm Hg. Systolic blood pressure in normal dogs when measured by oscillometric method has been reported to vary between 131 to 150 ± 20 mm Hg [
31]. Therefore 150 mm Hg appears a low cut off value to diagnose hypertension. When choosing a cut off value of 180 mm Hg, none of the dogs had episodes of hypertension. If based on the cutoff value of 150 mm Hg, there were several episodes of transient hypertension. This could very well be physiologic and related to stress [
30,
31]. Even in the event that these episodes of hypertension were related to the administration of taurolidine, the hypertension was transient and the dogs did not suffer any ill consequences in the short or long term. Hypotension has been defined as a mean arterial pressure <60 mmHg [
32‐
34]. Dog #2 had a transient episode of hypotension during the infusion (Figure
2).
Dogs with osteosarcoma
The most common side effect of taurolidine infusion in humans is a local reaction manifested by burning at the infusion site, numbness or soreness of the infusion arm, and erythematous striking at the IV site. Twenty-eight percent of subjects had facial flushing during the infusion and 5% each for headache, epistaxis, and nausea [
20]. None of the healthy dogs appeared to be bothered by the taurolidine infusion in their peripheral vein. The two dogs that received doxorubicin and taurolidine in the same peripheral vein during the same treatment sessions developed limb edema. One dog had a vasculopathy at necropsy but not the other. It was hypothesized after these 2 dogs that the administration of doxorubicin and taurolidine in the same vein caused too much irritation and therefore it was decided to administer the treatments through a central line. For this, a VAP was placed in the jugular vein in the following dogs except dog #13. However, because the VAP became non-functional in dog #12, the carboplatin and taurolidine were administered in different peripheral veins and the dog did well. Based on this, the carboplatin and taurolidine administrations were given in different peripheral veins at each session in dog #13 and this dog also did not show any adverse effects at the injection sites. We suspect that injecting the combination of doxorubicin and tauroldine is not feasible in the same peripheral vein, at least not on the same day. It is unknown if carboplatin and taurolidine can be injected in the same vein during the same treatment session.
Cytopenias experienced by dogs with osteosarcoma in this study were comparable to those reported with carboplatin alone. In a study of 65 dogs with osteosarcoma treated with carboplatin alone, 20% of the carboplatin cycles lead to neutropenia grade I, 3% to grade II, and 1% to grade III; Eleven percent of the carboplatin cycles lead to thrombocytopenia grade I, 12% to grade II, and 4% grade III [
23]. In this study, 7 episodes of neutropenia and one episode of thrombocytopenia were recorded after 18 combined doses of taurolidine and carboplatin. None of the cytopenias in this study were life-threatening as 5 neutropenic episodes were grade I and 2 were grade II and the one thrombocytopenic episode was grade II. Moreover, 3 out 5 doses of carboplatin administered alone resulted in grade 1 neutropenia. It is possible that the cytopenias in this study were underestimated because of the timing of the blood draws after a treatment. The nadir neutrophil and platelet counts occur on day 14 in dogs receiving carboplatin alone [
35]. In our study, when the blood draws were done on days 7–13, it is possible that the neutrophil and platelet counts were normal but later dropped below normal levels. In another study 76% of the neutropenic episodes were recorded between days 18 and 22 after a carboplatin treatment [
23]. In our study 4 of the 7 neutropenic episodes were recorded 21 to 29 days post taurolidine and carboplatin treatment.
In one study 24% of dogs with osteosarcoma experienced at least one episode of gastrointestinal toxicity with carboplatin alone [
36]. In another, 4% of the cycles of carboplatin resulted in grade I vomiting, 2% in grade II, and <1% in grade III. Five percent of the cycles of carboplatin resulted in grade I diarrhea, 2% in grade II, and <1% in grade III [
23]. The gastrointestinal toxicity experienced in the clinical dogs in this study compares favorably with these numbers.
Cardiac toxicity is a well-known and documented side effect of doxorubicin. The toxicity is cumulative [
37]. In one of the largest studies in dogs reporting on the use of doxorubicin for the treatment of osteosarcoma, 8% developed heart disease [
22]. In another recent study of 94 dogs with various neoplasms treated with doxorubicin, the incidence of cardiotoxicity was 8% [
38]. The dog in our study that developed congestive heart failure was an Irish Wolfhound and the cardiac failure was clinically diagnosed after a cumulative dose of 90 mg/m
2 of doxorubicin. Irish Wolfhound is a breed predisposed to developing dilated cardiomyopathy with a prevalence to be reported as high as 24% [
39]. In the case in this study, a complete evaluation of the heart to include ECG and echocardiogram before starting the combination treatments was performed and found to be completely normal. Dilated cardiomyopathy was diagnosed at necropsy. It is not possible to tell if the dilated cardiomyopathy was a result of doxorubicin toxicity or idiopathic in an Irish wolfhound. On one hand the time frame to develop the dilated cardiomyopathy with congestive heart failure seems short (6 weeks from initiating doxorubicin therapy) but on the other hand the cumulative dose of doxorubicin (90 mg/m2) was relatively low for cardiotoxicity. It may very well be a combination of using doxorubicin in a high risk breed. Whether the taurolidine played any role in promoting the cardiotoxicity of doxorubicin is unknown but must be considered.
Renal toxicity caused by doxorubicin is known to occur in rabbits, pigs, and cats but appears to be very rare in dogs [
37,
40,
41]. Rabbits treated with doxorubicin developed azotemia, proteinuria, and hypoproteinemia [
42]. In this study, two dogs that were administered doxorubicin with taurolidine developed nephropathies (dogs #8 and 9). One dog developed proteinuria and hypoalbuminemia. The other dog developed renal insufficiency. In the dog with renal insufficiency, the damage was reversible as the ability to concentrate recovered. The first dog had a necropsy which revealed severe thickening of the Bowman’s capsule, mesangial matrix, and the presence of occasional proteinaceous casts in the tubules. Reported changes with doxorubicin alone in cats, rabbits, and pigs include tubular casts, tubular dilation, stromal sclerosis, glomerular sclerosis and vacuoles, thickening of Bowman’s capsule, and thickened mesangial matrix [
40,
41,
43]. The mechanism of nephrotoxicity with doxorubicin remains unknown [
42]. Given that two of the 3 dogs that received doxorubicin with the taurolidine developed nephropathies and this is rare in dogs with doxorubicin alone, the possibility exists that taurolidine may potentiate the nephrotoxic effects of doxorubicin in this species.
To the authors’ knowledge, ototoxicity has not been reported with carboplatin in dogs. In children, ototoxicity has been reported in 4 to 10% of patients after receiving carboplatin [
44,
45]. It is possible that ototoxicity in the form of hearing loss occurs more frequently than we realize in dogs given the difficulty to assess this function in this species [
46]. But given the rarity of ototoxicity of carboplatin in dogs, it is possible that taurolidine had a role in potentiating in dog #11 this known toxicity in humans.
Although not a goal of this study, 1 of the 5 dogs that received carboplatin in combination with taurolidine was alive at the time of writing, 1186 days postoperatively, with bone metastases diagnosed 1071 days postoperatively (dog #9) (Table
2). One dog (#10) died 897 days post amputation with pulmonary metastasis. This dog also received another investigational drug starting 270 days post amputation. The other 3 dogs were euthanized 174 (dog #11), 163 (dog #12), and 116 (dog #13) days post removal of the tumor. All 3 dogs euthanized had one or more negative prognostic factors. In dog #11, the tumor was located in the proximal humerus, the anatomic location with poor prognosis in the appendicular skeleton [
36,
47]. Dog #12 had a tumor that involved 82% of the length of the radius and a high mitotic index (over 21/3 high power fields [HPF]). Dog #13 had a large tumor volume with a high mitotic index (15/3 HPF). Large volume [
48,
49], percent of radius affected (>29% affected) [
50], and mitotic index (>5/3 HPF) [
22,
23,
51] have been reported as negative prognostic factors for disease-free interval or survival. In 2 studies on prognosis when using carboplatin alone in dogs with OS, the median disease-free interval was 256 and 137 days and median survival was and 307 and 277 days [
23,
36].
A recent study found that taurolidine enhanced metastasis of osteosarcoma to the liver and lungs in mice and was toxic to the liver [
52]. In the present study, there is no evidence of taurolidine being toxic to the liver. One dog with OS (#12) showed an elevation in alanine transaminase (ALT) after the 4th taurolidine treatment and 6th carboplatin treatment (371 IU/L [normal range 0–113 IU/L). Two weeks later it was down to 129 IU/L. Two dogs with OS (#9 and 11) had an elevated alkaline phosphatase (ALP) or ALT before receiving taurolidine which returned to normal after receiving the combination treatments. Four healthy dogs (#1, 3, 4, and 6) had an elevated liver parameter (either ALP, ALT, or gamma-glutamyl transpeptidase [GGT], or combination of) before receiving taurolidine and the parameters either returned to normal or decreased after the taurolidine infusion. Furthermore, in the 3 dogs where a necropsy was performed, there was no evidence of liver damage. The doses of taurolidine that caused liver damage in mice were 750 mg/kg and 500 mg/kg. Based on allometric scaling of cancer drugs proposed by Freireich, et al. (1966) the equivalent dose of a drug in a dog is 1/6 the dose in a mouse [
53]. This translates to the equivalent of 125 mg/kg and 83 mg/kg in a dog. The dose used in our study (150 mg/kg) was higher than the one used in the study in mice if proportions are maintained across species.
It is not possible to tell if taurolidine enhanced metastasis of osteosarcoma to the lungs and liver in the dogs of this study. Metastases to the lungs are the most common with osteosarcoma and are present in over 90% of dogs over the course of the disease [
2]. In this study, two dogs died with pulmonary metastasis following treatment with taurolidine. Metastasis to the liver has been reported in dogs [
54]. In this study one dog was found to have metastasis to the liver on necropsy. An important distinction between this study and the one in mice where taurolidine enhanced the pulmonary and liver metastasis is that dogs in our study were also administered either doxorubicin or carboplatin.