High-flow isolation perfusion of the rat hind limb in vivo.

An in vivo model for perfusion chemotherapy of the rat hind limb is described in which perfusion at a predetermined flow rate of up to 4 ml.min-1 can be rapidly established in a tourniquet-isolated limb. Standard additions to the semisynthetic perfusion medium included glucose, insulin, and lactate from fresh human erythrocytes. Arteriovenous differences in oxygen content revealed a gradual increase with time of the rate of oxygen consumption until a plateau phase was reached. The plateau rates of oxygen consumption, and of utilization of lactate and of acetoacetate, when supplied, were significantly higher at 4 ml flow.min-1 than at 1.2 ml.min-1. When perfusion was performed at 4 ml.min-1, and suitable metabolic substrates were supplied, the rate of oxygen consumption at the plateau level was apparently equal to the estimated in vivo rate.

Dosimetry in isolation perfusion of the limbs by assessment of perfused tissue volume and grading of toxic tissue reactions.

The optimal single dosage of melphalan in isolation perfusion of the limbs for malignant melanoma was assessed. For this purpose a method to determine the volume of the isolated region in the individual patient and a grading system for the reaction of the normal tissues were introduced. A strictly standardized pharmacosurgical routine was developed that permitted an analysis of the correlation between dosage and the grade of toxic reaction in 90 perfusions. The optimal dosage of a cytostatic drug was considered to be the highest amount tolerated at an acceptable risk. Melphalan at 10 mg/l perfused tissue was determined as the likely optimum. This dose provoked remarkably little variation in toxicity, all reactions falling within a safe range. No exception to the applicability of this dosage was encountered.

Effects of alkylating agents on the DNA replication of cultured Yoshida sarcoma cells.

Logarithmically growing Yoshida sarcoma cells were treated for 1 h with low (2 decades cell kill) or high (more than 6 decades cell kill) doses of alkylating agents. Pulse and chase labelled DNA from treated cells were studied by alkaline sucrose gradient centrifugation. Nitrogen mustard (HN-2), 4-hydroperoxycyclophosphamide (CY-OOH), melphalan (L-PAM) and chlorambucil (CA) had no effect on the elongation rate of newly replicated DNA, both at low and high doses, although per cell the rate of DNA synthesis declined as inferred from the rates of [3H]thymidine incorporation compared to the increase in numbers of S phase cells in the treated populations. It is concluded that these drugs act specifically on the initiation step of the DNA replication, leaving chain elongation undisturbed. At low doses the chemically related sulphur mustard (SM) had also no effect on the maturation of new DNA but at high doses a decreased elongation rate was observed. A transient inhibition of chain growth was observed following treatment with a low dose of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, the intercalating agent adriamycin showed a severe but delayed effect resulting in an almost complete block of the maturation.

Enhanced cytostatic effectiveness of aniline mustard against 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors during regression in response to ovariectomy.

Sprague-Dawley rats bearing 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors were treated with either of two aromatic alkylating agents, aniline mustard or melphalan, alone or combined with ovariectomy. Both drugs were applied once a week for 8 weeks. Eight-four percent of the tumors responded to ovariectomy, 38% regressing completely and 46% regressing partially. Aniline mustard, though virtually ineffective as a single agent, appeared synergistic with ovariectomy: a 100% regression rate (72% complete, 28% partial) was observed for this combination. Treatment with melphalan was as effective as ovariectomy, but the combination of melphalan with ovariectomy was no more effective than either treatment alone. The end product of aniline mustard metabolism, p-hydroxyaniline mustard O-glucuronide, may be more extensively activated by beta-glucuronidase in hormonally regressing than in growing or stationary tumors. Intratumoral levels of beta-glucoronidase occurring in DMBA-induced tumors 4 days after ovariectomy were found to be similar to those in the aniline mustard-sensitive mouse plasma cell tumor ADJ/PC6. It remains to be more extensively studied whether an effect of endocrine treatment on tumor beta-glucuronidase levels, and possibly on intracellular distribution of enzyme, could be used therapeutically. An effectively scheduled cytostatic treatment (with a drug conjugate such as that formed metabolically from aniline mustard) in conjunction with ovariectomy might be effective in the treatment of hormone-responsive breast cancer.

Effects of treatment with cyclophosphamide on hormone-dependent and hormone-independent tumor cells in transplanted GR mouse mammary tumors.

Previous studies have indicated that hormone-responsive mammary tumors of GR mice are mixed populations of hormone-dependent and autonomous cells. We have now investigated whether these two cell types differ in susceptibility to cytostatic treatment. Experiments in which cyclophosphamide was injected in tumor-bearing mice did not reveal significant differences in percentage of inhibition between hormone-dependent, hormone-responsive, and hormone-independent tumors. Furthermore, the estrogen and progesterone receptor contents of the residual tumor masses after cyclophosphamide treatment were about the same as those of untreated tumors. When the cytostatically treated tumors were transplanted, the degree of hormone responsiveness of the transplants did not differ from that of transplants derived from untreated tumors, nor did their hormone receptor contents. These results indicate that the hormone-dependent and autonomous cells of GR mouse mammary tumors are inhibited to similar extents by cyclophosphamide. The possible significance of these results for combined endocrine therapy and chemotherapy is discussed.

Two stable Fenton oxidation products of cyclophosphamide (NSC-26271) as precursors of 4-hydroxycyclophosphamide (NSC-196562) under physiologic conditions.

Cytostatic and chemical properties of two cyclophosphamide (CP) 4-peroxides, 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide anhydro-dimer, that were earlier isolated and identified after Fenton oxidation of CP, are summarized and discussed. Their cytostatic toxicities, which are quantitatively comparable to those of the primary metabolite of CP, 4-hydroxycyclophosphamide, are explained from their apparent spontaneous conversion to mono-oxidized forms of CP, both in vitro and in vivo. The conversion is shown to proceed via autocatalyzed reactions, which are thought to imply the occurrence of free radicals during deoxygenation. The point is raised whether any qualitative difference in cytotoxicity might be caused by the action of these free radicals. Current dose-effect studies of residual colony-forming ability after treatment of 3T6 mouse fibroblasts and Yoshida sarcoma cells in suspension culture are introduced. Further studies on the local toxic action of the radicals under in vivo conditions are necessary if the peroxides are to be introduced in local or regional clinical treatments.