P. Lelieveld

The effect of treatment in fractionated schedules with the combination of x-irradiation and six cytotoxic drugs on the RIF-1 tumor and normal mouse skin

RIF-1 tumors, implanted syngeneically in the gastrocnemius muscles of the right hind legs of C3H/Km mice, were treated either with X ray alone, drug alone, or drug and X ray combined. The drugs tested were bleomycin, BCNU, cis-diamminedichloro platinum, adriamycin, cyclophosphamide, and actinomycin-D. All drugs were administered either in the maximum tolerated dose or a dose that causes minimal tumor growth delay. Both drugs and X rays were administered either as a single dose or in five daily fractions. In addition to the single modality controls, seven different schedules of combined modalities were tested. Tumors were measured periodically after treatment in order that the day at which each tumor reached 4 times its initial cross-sectional area, i.e., its size at the time of treatment, could be determined. The effect of treatment on tumors was based upon excess growth delay (GD), i.e., T400% (treated)-T400% (untreated control). Treatment effects for the same combined modality schedules were also determined for normal skin, using the early skin reaction as an endpoint. Dose effect factors (DEF) were computed for all combined modality schedules and were based upon calculated radiation dose equivalents. We also calculated supra-additivity ratios, SRI and SRII, therapeutic gain factors and adjusted therapeutic gain factors. The only drugs to produce significant supra-additivity with X rays were cis-Pt and cyclo. Maximum supra-additivity for cis-Pt was afforded by divided doses of the drug (5 X 2.4 mg/kg/day) given immediately before X ray (5 X 1000 rad/day) on 5 consecutive days, although 3 other schedules also produced significant supra-additivity. Maximum supra-additivity for cyclo was seen for a single dose of 100 mg/kg followed 1 day later by a course of 5 daily X ray doses (5 X 1000 rad/day), and at least one other schedule produced almost as great an effect.

Preclinical studies on toxicity, antitumour activity and pharmacokinetics of cisplatin and three recently developed derivatives☆

Preclinical studies were performed in mice, rats and dogs of cis-diamminedichloroplatinum(II) (CDDP) and its derivatives cis-1,1-di(aminomethyl) cyclohexane platinum(II) sulphate (TNO-6), cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) (CBDCA) and cis-dichloro, trans-dihydroxybis-isopropylamine platinum(IV) (CHIP). In mice toxicity and antitumour activity were determined. All three derivatives were at least as toxic as CDDP for haemopoietic stem cells and were less active than CDDP against the mouse tumours leukaemia L1210 and osteosarcoma C22LR. Toxicology studies in rats revealed no renal toxicity after a single dose of TNO-6. Fractionated doses of TNO-6 and CBDCA did cause renal toxicity but less than CDDP. CHIP produced little or no kidney damage. In dogs, TNO-6 (1.5 mg/kg) produced more severe kidney damage--although this was reversible--than CDDP (2 mg/kg). Half-lives of distribution were 4.0-5.1 min for TNO-6 and 9.7 min for CDDP, while half-lives of elimination were 3.6-6.6 days and 5.9 days respectively. Plasma levels, normalized for the dose, were at least two times higher after TNO-6 than after CDDP. Twelve weeks after drug administration, plasma levels were undetectable, while tissue concentrations could still be measured. The platinum concentration in kidney cortex was higher after CDDP than after TNO-6.

Factors determining cell killing by chemotherapeutic agents in vivo—I.: Cyclophosphamide

Abstract The effect of various chemotherapeutic agents in vivo on the survival of mouse haemopoietic spleen colony-forming cells and osteosarcoma cells is compared and a detailed study is made of the dose-effect curve of cyclophosphamide for the haemopoietic stem cells in normal-mouse bone marrow and in rapidly repopulating spleen. Different parts of the dose-effect curve could be attributed to different mechanisms and it was evident that cyclophosphamide is more effective against rapidly proliferating cells. After elimination of an artefact caused by the assay technique it could be shown that the greater activity versus rapidly proliferating cells is not caused by a higher sensitivity when the cells are exposed in a particular phase of the mitotic cycle, as is the case for instance with vinblastine. The hypothesis is advanced that DNA alkylation by cyclophosphamide or its metabolites may take place in any part of the cell cycle, but that cell killing will occur only if the lesion has not been repaired before DNA synthesis takes place. Finally the results show that only part of the difference in activity of cyclophosphamide versus normal bone marrow stem cells and tumour cells may be ascribed to differences in the rate of cell proliferation; other discriminating mechanisms must be involved.

The Survival of Colony-forming Units in Mouse Bone-marrow after in Vivo Irradiation with D–T Neutrons, X- and Gamma-radiation

SummaryIn collaborative experiments the CFU survival in mouse bone-marrow has been investigated after in vivo irradiation with D–T neutrons, 300 kV x-rays and 60Co and 137Cs gamma-rays. All the survival curves for x- and gamma-radiation showed the same extrapolation number (2·47 ± 0·28), whereas D0 values of 72·2 ± 1·7 rads and 90·7 ± 2·4 rads, respectively, were found for the two types of radiation. With D–T neutrons, the Manchester group found a D0 value of 62·5 ± 2·2 rads; the Rijswijk group found a D0 value of 78·3 ± 2·1 rads. A common extrapolation number of 1·34 ± 0·16 could be calculated for the neutron survival curves. Owing to the difference in extrapolation numbers for x-rays and neutrons, the RBE depends on the level of survival. From the combined results of both groups a mean RBE of 1·1 can be derived for low surviving fractions.The 25 per cent difference in the effectiveness of D–T neutrons, as measured by the two groups, was not due to dose-rate nor to the strain of mice, and could only in p...

Factors determining cell killing by chemotherapeutic agents In Vivo—II.: Melphalan, chlorambucil and nitrogen mustard

The sensitivity of resting spleen colony-forming cells in normal mouse bone marrow and of rapidly-proliferating spleen colony-forming cells in repopulating mouse spleen to a number of bis-chloro-aethyl-amines was determined. The rapidly-growing cells were more sensitive to each of the agents than the resting cells. The equal degree of sensitization to these agents by an increased rate of proliferation of the cells points to a common mechanism for all agents of this type. It is suggested that repair of damaged DNA, by excision of alkylated bases or oligonucleotides before the damage becomes irreversible during the next DNA synthetic period, is the most likely explanation of the uniform difference in sensitivity caused by the modified rate of cell proliferation.

Response of a poorly reoxygenating mouse osteosarcoma to x-rays and fast neutrons.

Abstract A transplantable osteosarcoma, which had previously been shown to reoxygenate its anoxic cells very slowly after a single large dose of X-ray, was studied after X- and neutron-irradiation. It was shown to be relatively resistant to fractionated X-ray therapy, probably due to anoxia. Nevertheless it could be shown that a small degree of reoxygenation must also occur in this tumour. Its response to treatment with 15 MeV neutrons was unusual; RBE values for cell survival of 10 −3 were for fractionated irradiation 2·5 , for single dose in vivo irradiation 1·8 , and for in vitro irradiation 1·6 . The small difference between the RBE values for the well oxygenated osteosarcoma cells in vitro and for the partially hypoxic cells in vivo is associated with an unexpectedly high OER of 2·0 for neutron irradiation of these cells in vitro as compared with the OER for X-rays of 2·4 . The gain factor for neutrons, that is the ratio of OER values for X-rays and neutrons, is only 1·2 , indicating that although hypoxia may be a limiting factor for the cure of this tumour by fractionated X-ray theory, the use of neutrons does not offer a major advantage. The search for other models of radiotherapy-resistant tumours should be continued.

Comparison of metastatic disease after local tumour treatment with radiotherapy or surgery in various tumour models

Spontaneous metastases in lymph nodes and/or the lung were obtained after tumour cell inoculation of four mouse tumours and one rat tumour into the foot-pads of syngeneic animals or their F1 hybrids. Following local radiotherapy with doses of 45-80 Gy, significantly more mice died with metastases than following local amputation of the tumour-bearing foot when the 2661 carcinoma was involved. No significant difference was observed after these treatments for the other tumours. The enhancement of metastastic growth after local radiotherapy in the 2661 carcinoma seems not to be due to incomplete killing of tumour cells in the foot. The presence of irradiated normal structures and tumour tissue after radiotherapy promoted the outgrowth of 261 carcinoma cells which were outside the radiation field at the time of treatment. Evidently, even under similar experimental conditions, radiotherapy may enhance the growth of metastases from some tumours and not from others.

The effect of BCNU on mouse skin and spinal cord in single drug and radiation exposures

Abstract We set out to determine whether any interaction occurs between BCNU and radiation for the mouse skin and spinal cord. Single doses of BCNU of 10, 20, or 30 mg/kg were injected intraperitoneally as a function of time before or after irradiation of the foot or spinal cord of anesthesized C3H mice. Enhancement of the radiation skin reaction (dose enhancement factor=1.3) was seen when BCNU (30 mg/kg) was given 1 day, 6 hr, and 2 hr prior to irradiation of the foot with 2,500 rad, and a larger DEF of 1.6 was observed when BCNU was given immediately before the radiation dose. However, with a different mouse strain (BALB/c) not anesthetized at the time of irradiation, no significant enhancement following a dose of 20 mg/kg BCNU was observed. Experiments are in progress to determine the cause of these differences BCNU (10 mg/kg) was given 24 hr or immediately prior to various single doses of radiation to a 12 mm segment of the mouse spinal cord (T 11–12 to L 1–2 ), and the subsequent myelitis was scored monthly. The addition of BCNU to irradiation did not accelerate the development of myelitis, nor the ultimate proportion of animals developing hind limb paralysis: the 50% myelitis dose at 10 months (MD 50/10 mo ) values for irradiation alone, BCNU at the time of irradiation and 24 hr before were 3,722, 3,795 and 3,853 rad, respectively.

Experimental studies on the combination of radiation and chemotherapy.

Abstract Experimental studies were performed to evaluate the effect of approximately simultaneous application of radiation and a number of widely used cytostatic agents. Effects were tested in five systems: (a) Normal mouse haemopoietic spleen colony forming stem cells; (b) The same cells when induced to proliferate rapidly; (c) L1210 leukemia cells; (d) and (e) Microtumors in the lung, exposed to combined treatment at respectively 24 hr and 96 hr after intravenous inoculation of osteosarcoma cells in mice. Responses to combined treatment were different in the different test systems and so far no agent was identified which showed potentiation in all malignant cell systems tested. The difficulties in evaluating the results of simple tests are discussed.

Effectiveness of P-aminobenzyol-O-phenylenediamine (Goe 1734) against mouse, rat, and human tumour cells

SummaryThe new N-phenylbenzamide derivative Goe 1734 was tested for its antitumour effects against mouse, rat, and human tumours. The preparation showed marginal activity against leukaemia L1210, moderate activity against Lewis lung carcinoma, and high activity against osteosarcoma C22LR and Brown Norway myeloid leukaemia. In the subrenal capsule assay the drug was active against four (cisplatin: 2) of nine human tumours. An in vitro clonogenic assay did not reveal any activity of Goe 1734 when mouse osteosarcoma or human tumour cells were exposed for only 1 h. However, continuous exposure led to 70% or greater inhibition of colony formation at concentrations of 0.1–1 μg/ml (osteosarcoma) or 0.2–2 μg/ml (human tumours).