A pilot trial of doxorubicin containing phosphatidyldiglycerol based thermosensitive liposomes in spontaneous feline soft tissue sarcoma

Figures & data

Table 1. Overview of study protocol for the treatment of spontaneous feline soft tissue sarcoma with thermosensitive liposomal doxorubicin (DPPG₂-TSL-DOX) and local hyperthermia

	Initial exam	Treatment cycle 1	Treatment cycle 2–7	Recheck seven days after treatment	Final exam
DPPG2-TSL-DOX	–	X	x	–	–
Local hyperthermia	–	–	x	–	–
Pharmacokinetics	–	X	x	–	–
Tumour measurement
Calliper	x	X	x	x	x
c MRI or PET/MRI	x	–	–	–	x
Tumour biopsy	x	X	x	–	–
Physical examination	x	X	x	x	x
CBC	x	X	x	x	x
BC	x	X	x	x	x
Urinalysis	x	X	x	x	x
Echocardiography	x	–	–	–	x
Thoracic radiography	x	–	–	–	x
Abdominal ultrasound	x	–	–	–	x
Thyroxin	x	–	–	–	–
FIV/FeLV	x	–	–	–	–

Treatment cycles were performed every other week and under generalised anaesthesia.

Figure 1. Cat with huge spontaneous soft tissue sarcoma located at the back. After central insertion of a temperature probe, microwave hyperthermia with a tumour target temperature of 41.5 °C was applied for 60 min under general anaesthesia.

Table 2. Baseline characteristics of all cats with locally advanced soft tissue sarcoma assigned to treatment with liposomal doxorubicin (DPPG2-TSL-DOX) and simultaneously administered local hyperthermia.

Signalment						Tumour characteristics
No.	Breed	Age (years)	Sex	Weight (kg)	BCS	Site of tumour	Presentation	Volume* (cm^3)
# 1	DSH	8	fs	4.4	6/9	LR	P	260
# 2	DSH	11	fs	3.5	4.5/9	LR	P	42
# 3	DSH	11	mn	5.3	4.5/9	TW	R	128
# 4	DSH	12	mn	7.5	7.5/9	I	P	22
# 5	NFC	10	fs	3.9	4/9	TW	R	38
# 6	DSH	14	fs	5.6	7/9	I	P	30
# 7	DSH	12	mn	4.6	5/9	LR	R	17
# 8	DSH	12	fs	4.2	4.5/9	TW	R	4
# 9	DSH	13	mn	3.8	4.5/9	I	P	66
# 10	DSH	9	Fs	3.5	4.5/9	I	P	22
# 11	DSH	11	fs	5.9	7/9	TW	R	107

BCS: Body condition score; DSH: Domestic short hair; NFC: Norwegian forest cat; fs: female spayed; mn: male neutered; LR: Lumbar region; TW: Thoracic wall; I: Interscapular; P: Primary tumour; R: Recurrent disease.

* Manual calliper measurement.

Table 3. Toxicity data. Classification and grading of adverse events.

		Group I (N = 5)				Group II (N = 3)				Group III (N = 3)
Systemic toxicity	All events	G 1	G 2	G 3	G 4	G 1	G 2	G 3	G 4	G 1	G 2	G 3	G 4
Hematological
Neutropenia	1	–	–	–	–	1	–	–	–	–	–	–	–
Thrombocytopenia	4	1	–	1	–	–	–	–	–	2	–	–	–
Anaemia	8	1	3	–	–	2	1	–	–	–	1	–	–
Non-hematological
Weight loss	6	–	2	–	–	1	–	1	–	2	–	–	–
Anorexia	3	–	1	–	–	1	–	–	–	1	–	–	–
Vomiting	–	–	–	–	–	–	–	–	–	–	–	–	–
Nephrotoxicity	–	–	–	–	–	–	–	–	–	–	–	–	–
Hepatotoxicity	–	–	–	–	–	–	–	–	–	–	–	–	–
Cardiotoxicity	–	–	–	–	–	–	–	–	–	–	–	–	–
Immunologic effect
Hypersensitivity	–	–	–	–	–	–	–	–	–	–	–	–	–
Local toxicity related to hyperthermia
Skin	5	2*	–	1		–	–	1*		1	–	–

Cats with locally advanced soft tissue sarcoma were simultaneously treated with thermosensitive liposomal doxorubicin (DPPG₂-TSL-DOX) and local hyperthermia. All events, including non-related toxicities are listed. Treatment related side effects are highlighted with “*”.

Classification of adverse events were evaluated according to VCOG-CTCAE V1.1 (grade 1–4) and VRTOG acute radiation morbidity scheme (grade 1–3). Treatment group I was treated with escalating doses of 0.1–0.4 mg/kg DPPG₂-TSL-DOX and local hyperthermia. Group II received 0.4 mg/kg and group III received 0.6 mg/kg DPPG₂-TSL-DOX and local hyperthermia. G: Grade.

Table 4. Intratumoral temperature values of cats with locally advanced soft tissue sarcoma treated with liposomal doxorubicin (DPPG₂-TSL-DOX) and simultaneously administered hyperthermia.

Cycle	B		C		D		E		F		G
Temperature [°C]	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Mean	41.3	0.5	41.4	0.8	42.2	1.1	41.7	0.7	41.4	1.5	42.3	1.3
T90	40.7	1.2	40.4	1.3	40.9	0.9	40.7	0.8	39.9	2.4	41.5	1.3
T50	41.5	0.5	41.5	0.8	42.5	1.4	41.9	0.7	41.6	1.5	42.4	1.2
Tmax	42.9	1.3	43.0	1.0	44.6	2.4	43.7	2.0	43.4	1.8	44.5	2.6
Tmin	39.0	2.0	38.1	2.9	39.1	1.7	38.1	2.4	37.7	2.3	39.7	1.9
N	11		9		8		7		7		7

Intratumoral temperature values of treatment cycles B–G are listed. Cycle A was without HT. Displayed are the mean, median (T₅₀), the temperature level above which 90% of the data was recorded (T₉₀), maximum and minimum temperature in each cycle. Values shown are averaged over all cats # 1–11. SD denotes standard deviation and N number of animals treated in this cycle

Figure 2. Pharmacokinetic profiles of DOX after application of DPPG₂-TSL-DOX. (A) DOX plasma concentration in cats receiving no local hyperthermia treatment. A huge variation in the plasma concentration of DOX was observed in the lower dose level (0.1 mg/kg), that was not observed in the higher dose level (0.4 mg/kg). (B) DOX plasma concentration in one representative cat treated without (open diamonds) local hyperthermia in the first treatment cycle and with (closed diamonds) local hyperthermia in the second cycle.

Table 5. Pharmacokinetic parameters of doxorubicin encapsulated into DPPG₂-TSL obtained from cats with locally advanced soft tissue sarcoma.

	Without HT					With HT (0 – 60min)
Dosage(mg/kg)	N	Cmax (μg/ml)	%ID15min	tα (min)	AUC1–135 min(μg/ml*min)	N	Cmax(μg/ml)	%ID15 min	tα (min)	AUC1-135min(μg/ml*min)
0.1	5	0.9 (±0.5)	37.1 (±19.1)	22.5 (±13.2)	44 (±32)	9	0.8 (±0.5)	34.0 (±18.1)	10.3 (±5.1)	33 (± 23)
0.2	0	–	–	–	–	5	2.8 (±1.1)	55.6 (±21.6)	13.0 (±3.8)	140 (± 87)
0.4	3	6.4 (±1.8)	64.1 (±18.2)	39.5 (±9.9)	377 (±132)	12	8.8 (±3.9)	88.1 (±38.9)	25.1 (±24.8)	562 (± 352)
0.6	0	–	–	–	–	3	8.8 (±5.6)	58.2 (±37.5)	14.6 (±7.8)	404 (± 274)

Cats were treated with four different dose levels of DPPG₂-TSL-DOX and with or without local hyperthermia.

C_max, maximum serum concentration of DOX; %ID_15min, percentage of the (theoretical) maximum concentration of DOX injected to cats measured after completion of the infusion of DPPG₂-TSL-DOX; t_α, initial (alpha) plasma half-life of DOX; AUC_{1–135 min}, area under the curve calculated from t = 1 to 135 min. The standard deviation is given in parentheses.

Table 6. Tumour response in cats with locally advanced soft tissue sarcoma treated with thermosensitive liposomal doxorubicin (DPPG₂-TSL-DOX) at four different dose levels and simultaneous local hyperthermia.

(A) Tumour volume was determined by manual calliper measurements.
		Tumour volume (cm^3)
Treatment group	No.	Initial	After treatment	Change (%)	Response evaluation
I	# 1	260	240	−8	SD
	# 2	42	–	–	–
	# 3	128	–	–	–
	# 4	22	14	−53	SD
	# 5	38	–	–	–
II	# 6	30	23	−24	SD
	# 7	17	8	−46	SD
	# 8	4	–	–	–
III	# 9	66	37	−38	SD
	# 10	22	5	−79	PR
	# 11	107	293	+174	PD

Table

(B) Tumour volume was determined by MRI and PET/MRI measurements.
		Tumour volume (cm^3)			Metabolic tumour response according to PERCIST			Response evaluation
Treatmentgroup	No.	Initial	Aftertreatment	Change (%)	Initial SUVpeak mean	SUVpeak meanafter treatment	Change (%)	Volume	PERCIST(SUVmax)
I	# 1	318	434	+37	–	–	–	SD	–
	# 2	51	–	–	–	–	–	–	–
	# 3	99	–	–	–	–	–	–	–
	# 4	20	100	+391	–	–	–	PD	–
	# 5	No MRI	–	–	–	–	–	–	–
II	# 6	34	50	+ 47	–	–	–	PD	–
	# 7	17	17	0	–	–	–	SD	–
	# 8	4	–	–	–	–	–	–	–
III	# 9	66	138	+108	6.4	3.1	−31	PD	MPR
	# 10	52	16	−69	8.8	5.1	−34	PR	MPR
	# 11	118	389	+230	8.9	8.0	−11	PD	MSD

Volumetry: Tumour volume is calculated by following formula: V = (A × B×C×π)/6. PR: ≥65% decrease in tumour volume; SD: <65% decrease and <44% increase in tumour volume; PD: ≥ 44% increase in tumour volume.

PERCIST: MPR: metabolic partial response, MSD: metabolic stable disease.

Treatment group I was treated with escalating doses of 0.1–0.4 mg/kg DPPG₂-TSL-DOX and local hyperthermia. Group II received 0.4 mg/kg and group III received 0.6 mg/kg DPPG₂-TSL-DOX and hyperthermia.

Tumour volume was calculated by V = (A × B×C×π)/6. PR: ≥65% decrease in tumour volume; SD: < 65% decrease and <44% increase in tumour volume; PD: ≥ 44% increase in tumour volume.

Treatment group I was treated with escalating doses of 0.1–0.4 mg/kg DPPG₂-TSL-DOX and local hyperthermia. Group II received 0.4 mg/kg and group III received 0.6 mg/kg DPPG₂-TSL-DOX and hyperthermia.

Figure 3. (A) # 10: PET/MRI fusion images demonstrating tumour response after two and after six treatments with DOX 0.6 mg/kg in DPPG₂-TSL-DOX. (B) Macroscopic view and microscopic pictures (HE, ×50 magnification) of the subsequently resected tumour with complete necrosis and chronic inflammation (above) and residual malignant spindle cells (bottom). Complete tumour necrosis in the tumour part near to the applicator indicating insufficient heating in the basal part.