Usefulness of combined treatment with continuous administration of tirapazamine and mild temperature hyperthermia in γ-ray irradiation in terms of local tumour response and lung metastatic potential

Figures & data

Table I.  Surviving fractions and micronucleus frequencies at 0 Gy.

Treatment	Total cells	Quiescent cells
Plating efficiency (%)
Without TPZ or MTH	84.4 ± 8.2^a	–
With TPZ (i.p.*)	65.7 ± 6.6^b	–
With TPZ (cont.**)	37.0 ± 3.5^c	–
With TPZ (i.p.) and MTH	57.6 ± 5.8^d	–
With TPZ (cont.) and MTH	31.4 ± 2.1^e	–
Micronucleus frequency
Without TPZ or MTH	0.050 ± 0.007^f	0.077 ± 0.008^k
With TPZ (i.p.)	0.104 ± 0.010^g	0.129 ± 0.013^l
With TPZ (cont.)	0.147 ± 0.015^h	0.181 ± 0.017^m
With TPZ (i.p.) and MTH	0.128 ± 0.012^i	0.163 ± 0.016^n
With TPZ (cont.) and MTH	0.170 ± 0.019^j	0.212 ± 0.020^o

TPZ, tirapazamine; MTH, mild temperature hyperthermia.

*Single intraperitoneal administration.

**Continuous subcutaneous administration.

Values are presented as mean ± standard error (n = 9). Micronucleus frequencies for quiescent cells were significantly larger than those for total cells under all treatment conditions (p < 0.05). In addition, in each following combination of two values, there was the significant difference between the two values (p < 0.05); a and (b, c, d or e), f and (g, h, i or j), k and (l, m, n or o), b and c, d and e, g and h, i and j, l and m, n and o.

Figure 1. Cell survival curves for the total cell population from B16-BL6 tumours irradiated with γ-rays following the single intraperitoneal (i.p.) or continuous subcutaneous (cont.) administration of tirapazamine (TPZ) in combination with mild temperature hyperthermia (MTH) on day 18 after tumour cell inoculation. ○ γ-ray irradiation only; Δ γ-ray irradiation after single intraperitoneal administration of TPZ; ▴ γ-ray irradiation after single intraperitoneal administration of TPZ with MTH; □ γ-ray irradiation after continuous subcutaneous administration of TPZ; ▪ γ-ray irradiation after continuous subcutaneous administration of TPZ with MTH. Bars represent standard errors (n = 9).

Figure 2. Dose response curves of the net micronucleus frequency for total (left panel) and quiescent (right panel) cell populations from B16-BL6 tumours irradiated with γ-rays following the single intraperitoneal (i.p.) or continuous subcutaneous (cont.) administration of tirapazamine (TPZ) in combination with mild temperature hyperthermia (MTH) on day 18 after tumour cell inoculation. ○ γ-ray irradiation only; △ γ-ray irradiation after single intraperitoneal administration of TPZ; ▴ γ-ray irradiation after single intraperitoneal administration of TPZ with MTH; □ γ-ray irradiation after continuous subcutaneous administration of TPZ; ▪ γ-ray irradiation after continuous subcutaneous administration of TPZ with MTH. Bars represent standard errors (n = 9).

Table II.  The effect* of tirapazamine on each end point.

Type of administration	Total cells	Quiescent cells
Surviving fraction = 0.03
Single intraperitoneal	1.2 ± 0.1^a	–
Continuous subcutaneous	1.5 ± 0.15^a	–
Net micronucleus frequency = 0.6
Single intraperitoneal	1.1 ± 0.05^b,d	1.3 ± 0.1^c,d
Continuous subcutaneous	1.3 ± 0.1^b,e	1.55 ± 0.15^c,e

*The dose of radiation required to obtain each end-point without each treatment in relation to that required to obtain each endpoint with each treatment.

^a-eThe significant differences between two values (p < 0.05). Values are presented as mean ± standard error (n = 9).

Table III.  The effect* of mild temperature hyperthermia on each end point.

Type of tirapazamine administered	Total cells	Quiescent cells
Surviving fraction = 0.03
Single intraperitoneal	1.1 ± 0.05	–
Continuous subcutaneous	1.05 ± 0.05	–
Net micronucleus frequency = 0.6
Single intraperitoneal	1.1 ± 0.05	1.15 ± 0.05
Continuous subcutaneous	1.05 ± 0.05	1.1 ± 0.05

*The ratio of the dose of radiation necessary to obtain each end point without mild temperature hyperthermia to that needed to obtain each end point with mild temperature hyperthermia.

Values are presented as mean ± standard error (n = 9).

Table IV.  Dose-modifying factors for quiescent cells relative to the total cell population.*

Type of tirapazamine administered	Without MTH	With MTH
Net micronucleus frequency = 0.6
Without TPZ	1.65 ± 0.15^a,b	–
Single intraperitoneal	1.4 ± 0.1^a	1.35 ± 0.1
Continuous subcutaneous	1.45 ± 0.1^b	1.4 ± 0.1

MTH, mild temperature hyperthermia.

*The ratio of the dose of radiation necessary to obtain each end point in the quiescent cell population to that needed to obtain each end point in the total tumour cell population.

^a,^bThe significant differences between two values (p < 0.05). Values are presented as mean ± standard error (n = 9).

Figure 3. Tumour growth curves for B16-BL6 tumours with (solid lines) or without (dotted lines) γ-ray irradiation at a dose of 16 Gy following the single intraperitoneal (i.p.) or continuous subcutaneous (cont.) administration of tirapazamine (TPZ) in combination with mild temperature hyperthermia (MTH) on day 18 after tumour cell inoculation. ○ γ-ray irradiation only; △ γ-ray irradiation aftersingle intraperitoneal administration of TPZ; ▴ γ-ray irradiation after single intraperitoneal administration of TPZ with MTH; □ γ-ray irradiation after continuous subcutaneous administration of TPZ; ▪ γ-ray irradiation after continuous subcutaneous administration of TPZ with MTH. Bars represent standard errors (n = 9).

Table V.  The period (days) required for each tumour to become twice as large as on day 18 after tumour cell inoculation.

Type of tirapazamine administered	Without MTH	With MTH
Without irradiation
Without tirapazamine	2.2 ± 0.3^a,b	–
Single intraperitoneal	3.8 ± 0.4^a,c	4.2 ± 0.4^d
Continuous subcutaneous	4.7 ± 0.5^b,c	5.4 ± 0.5^d
With irradiation at a dose of 16 Gy
Without tirapazamine	11.0 ± 1.2^e,f	–
Single intraperitoneal	16.5 ± 1.7^e,g	19.4 ± 2.0^h
Continuous subcutaneous	22.0 ± 2.4^f,g	24.2 ± 2.5^h

MTH, mild temperature hyperthermia.

^a-hThe significant differences between two values (p < 0.05). Values are presented as mean ± standard error (n = 9). Values for irradiation at the dose of 16 Gy were significantly larger than those for no irradiation under all treatment conditions (p < 0.05).

Figure 4. Counted numbers of macroscopic metastases in the lung on day 35 after tumour cell inoculation as a function of the dose of γ-ray irradiation following the single intraperitoneal (i.p.) or continuous subcutaneous (cont.) administration of tirapazamine (TPZ) in combination with mild temperature hyperthermia (MTH) on day 18 after tumour cell inoculation. ○ γ-ray irradiation only; △ γ-ray irradiation after single intraperitoneal administration of TPZ; ▴ γ-ray irradiation after single intraperitoneal administration of TPZ with MTH; □ γ-ray irradiation after continuous subcutaneous administration of TPZ; ▪ γ-ray irradiation after continuous subcutaneous administration of TPZ with MTH. Bars represent standard errors (n = 9).

Table VI.  The numbers of metastases from the irradiated tumours that received cytotoxic treatment producing a similar initial local effect.*

Type of tirapazamine administered	Without MTH	With MTH
Surviving fraction = 0.03
Without tirapazamine	14.2 ± 1.4^a,b	–
Single intraperitoneal	13.0 ± 1.3	12.2 ± 1.2
Continuous subcutaneous	11.4 ± 1.1^a	10.8 ± 1.1^b

MTH, mild temperature hyperthermia.

*Based on the data shown in Figure 4, the estimated numbers of lung metastatic nodules from local tumours that received γ-ray irradiation with or without tirapazamine in combination with mild temperature hyperthermia, which produced an identical surviving fraction of 0.03 as an initial effect in Figure 1.

^a,^bThe significant differences between two values (p < 0.05). Values are presented as mean ± standard error (n = 9).