Je souhaite la bienvenue, et j'adresse mes remerciements, à tous les chercheurs et savants, venus de diverses régions d'Europe et d'Amérique et qui, aujourd'hui, se trouvent réunis dans notre Institut pour discuter des problèmes qui ne peuvent pas être plus actuels, concernant les recherches sur les antimitotiques.

Je remercie les Autorités qui ont bien voulu donner leur adhésion à ce Symposium et ont voulu l'honorer de leur présence.

Notre réunion prend une signification particulière du fait que les études sur les antimitotiques à régard de la chimiothérapie expérimentale des tumeurs ne remontent pratiquement, de façon officielle, qu'à une vingtaine d'années. C'est en effet en 1946 que parurent les travaux de Haddow et Sexton sur « Influence of carbamic esters (uretanes) on experimental animal tumors » (Nature, 157, 1946) et ceux de Gilman et Philips sur « The biological actions and the therapeutic applications of the o-chlo-roethyl-amines and sulphides » (Science, 103, 1946). Ce n'est que deux années plus tard que paraîtront les observations de Sidney Farber et de ses collaborateurs sur l'emploi des antifoliques dans la leucémie aiguë des enfants. Au cours des années successives, noue assistons à une floraison de recherches sur les antimitotiques, spécialement à Londres, au Chester Beatty, à New York, au Sloan-Kettering, et à Boston, au Children's Cancer Research Foundation. Je ne rappellerai que les étapes les plus importantes de ces recherches qui ont conduit à une sélection de substances antimitotiques d'une efficace action antitumorale: en 1953, apparaissent la 6-mercaptopurine, le Colcemid, le dyméthansulphoniloxibutane; en 1956, le Chlorambucil; en 1960, le Trenimon, l'Endoxan, la Vinblastine, le 5-Fluorouracil; en 1962, la Vincristine et l'hydroxyurée; en 1963, les méthylidrazines. Et ce ne sont là que quelques uns des nombreux cytostatiques étudiés jusqu'à présent.

The cytotoxic effects of hydroxyurea and of related hydroxamic acid derivatives in vivo are briefly described. They occur selectively in tissues with high rates of cell renewal and they are of brief duration. Tissue concentration of hydroxyurea diminish rapidly as the result of renal excretion and metabolism; there is a close temporal relation between the physiological disposition of the agents and the cytotoxic changes. Hydroxyurea induces an immediate inhibition of DNA synthesis in proliferating tisdsues such as thymus, small intestine, and regenerating liver. Autoradiographic studies of mouse duodenum using tritiated thymidine have shown that the lethal susceptibility to hydroxyurea is restricted to cells in the S-phase of the mitotic cycles. Cells in G1, G2, and M are not damaged by the agent.

Chemotherapy of murine Rauscher virus-induced leukemia using Trenimon, 6-Mercaptopurine, 5-Fluorouracil and Melphalan is described. Therapy is based on nucleated cell counts in peripheral blood and body weight of the animals. Remarkable remissions of the number of nucleated cells — not occurring in untreated animals — are induced by Trenimon and 6-Mercaptopurine. Prolongation of survival times exceeds 100% in the groups treated with Trenimon, 6-Mercaptopurine, and 5-Fluorouracil.

Selected subjects have been treated with cyclophosphamide and nitrogen mustard. The granulocytopenia has been followed by repeated in vivo labeling with DFP32 and the endotoxin test for evaluating the availability of the granulocyte reserve. The effect of steroid treatment on the recovery of the granulopoiesis has been studied with autotransfusions of in vitro DFP32 labeled granulocytes in the same subject and performed before, during and after the treatment was discontinued.

The following conclusions have been reached:

1. The efficiency of the granulopoiesis is based upon the availability of the bone marrow granulocyte reserve.

2. The bone marrow granulocyte mobilization with endotoxin and the in vivo granulocyte labeling with DFP32 give an evaluable information about the bone marrow granulocyte reserve.

3. The granulocytopenia due to antiblastic therapy corresponds to a depletion of the bone marrow granulocyte reserve.

4. The recovery of a “normal granulocyte count” preceeds the rebuilt of a “normal availability” of the bone marrow granulocyte reserve.

5. The recovery of the blood granulocyte count after prednison is not associated with any favourable change of the granulopoiesis.

The proliferative characteristics of human acute leukaemia cells are reported and the relationships between the proliferative alterations and differentiation defect of these cells discussed. The proliferative activity of acute leukaemia cells was also studied in relation to cytostatic treatment.

Emphasis is laid on the fact that in all cases of acute leukaemia the characteristic blast cells of the disease do not constitute a homogenous cell population but can be divided into various sub-classes with different kinetic and proliferative characteristics. It is also pointed out that all cytostatic treatment acts on the most actively proliferating classes and only indirectly on the non-proliferating classes.

Finally, the need for more detailed study of DNA synthesis at chromosome and sub-chromosome level for the purpose of more fully understanding the response of leukaemic cells to the various chemiotherapies is underlined.

The mode of action of the alkylating agent Melphalan on Yoshida ascites tumours resistant and sensitive to alkylating agents in rats has been studied. A combination of autoradiography and analytical histochemistry has been used to measure changes in the cell kinetics. The sensitive strain showed a marked fall in mitotic index, build up in G2 and finally interphase death occurring 24-72 hours after treatment. The resistant form showed only slight mitotic inhibition and a possible recovery phase 24-30 hours after treatment, without evidence of cytological damage.

Attempts to induce greater relative tumour specificity in cancer chemotherapeutic drugs may be aimed at (1) reducing toxicity towards host tissues or (2) increasing the susceptibility of the tumor.

1. Pretreatment with certain thiol derivatives has some protective action against the host toxicity of X-irradiation and of certain alkylating agents. However, in many cases even better protection against the tumour-inhibitory action occurs so that the therapeutic index is unfavourably affected.

2. Pretreatment with glucose which accentuates the pH difference between normal and tumour tissues can, in some cases, produce an increase in therapeutic index.

3. The most encouraging aspect is to explore and take advantage of biochemical variations from the normal tissue pattern which are found in certain tumours, e.g. the successful treatment of the ADJ/PC5 mouse myeloma with «aniline mustard» is believed to be related to the very high glucuronidase activity of this tumour.

This paper summarizes some studies recently carried out in this Institute in the field of cancer chemotherapy.

Three main groups of data are reported:

a) different sensitivity to chemotherapy of the same tumor transplanted in different sites of the body;

b) a method to study cancer dissemination as a basis for cancer chemotherapy;

c) a system to perfuse tissue culture with the blood of living animals as an approach to study duration of action of antitumoral drugs.

Molecular carriers with alkylating sites and sites endowed with antimetabolic activity were synthesized.

Phenylalanine and hydroxyphenylalanine having a dichlorodiethylamino group in the benzene ring were chosen as alkylating agents because of their capability to form peptides with natural and antagonist aminoacids.

The chemotherapeutic screening was carried out according to CCNSG rules with the only variant that the determination of the weight of the tumors of the treated mice was carried out on the day subsequent to that indicated by the CCNSC, i.e. at the 9th day after tumor implantation. In this same day the spleen weight was determined also. On the previous day, the white cell count was carried out and in way it was allowed an evaluation of the hemotoxicity.

The optical isomerism of the compounds plays a fundamental rôle in the chemotherapeutic activity.

Examples of different isomers of the tripeptide phenylalanyl-ethionyl-m-sarco-lysine and of the tetrapeptide arginyl-p-fluorophenyl-alanyl-glycyl-m-sarcolysine, endowed with markedly different chemotherapeutic activities (from o to 90) in relation to the different stereochemical configuration are reported.

Oxidative phosphorylation, ATPase, glycolysis, Nadase, succinic dehydrogenase, cytochrome-oxidase, glutamic dehydrogenase, lactic dehydrogenase amino acid oxidase, proteolysis were studied in biochemical pharmacology investigations.

The active compounds bring the phosphorylation ratio to zero in Sarcoma 180 mitochondria at doses reducing the P/O by 40-50% in normal liver mitochondria.

Interesting observations are pointed out for other enzyme systems, showing the specificity of the activity of some peptides at the biochemical level on tumor enzyme systems in comparison with the same enzyme systems of normal tissues.

An evaluation of the antimitotic activity of a group of antitumoral agents was performed. Their action was first assayed on chick embryo blood forming tissue, determining its survival, mitotic index and mitotic differential count: the statistically most active compounds were demecolcin, vinblastine and cyclophosphamide.

The same compounds were also tested on the in vitro blastic transformation of human leukocytes and their incorporation of tritiated DNA and RNA precursors.

The most active compounds in this assay was 5 fluorouracil; a lesser degree of inhibition was caused by vinblastine, cyclophosphamide and mechloretamine.

The effect of a single massive dose of the same compounds in vivo was also studied determining the in vitro activity of leukocytes of such patients, removed 48 hrs after the administration.

Preliminary results show that such leukocytes lose in variable degree their phyto-responsiveness in vitro.

Antimitotic agents induce pathological alterations by suspension of the normal cellular renewal: it results the picture of the so called « aregenerative pathology ». Moreover, they induce cytological abnormalities, like cellular vacuolization, alterations of the enzymatic cellular pattern, cellular giantism, etc. These alterations constitute the « cytological dysplasia by antimitotic drugs ». They are easily recognizable in many organs, i.e. in the haemopoietic system, lungs and pancreas. Particularly evident is the cellular giantism in the labial epithelia: in some of these cells of gynecologically normal women two Barr bodies are observed.

Daunomycin (Da) a metabolite of Streptomyces peucetius has a cytotoxic and antimitotic activity on normal and neoplastic mammalian cells grown in vitro and strongly inhibits a variety of experimental tumors.

The cell damage induced by Da is mainly nuclear.

A finely granular appearance of the chromatin and marked alterations in shape and size of nucleoli in resting cells and many mitotic aberrations in mitotic cells were observed.

Biochemical and autoradiographic researches show that the binding of Da with DNA causes a reduction of the DNA and RNA synthesis, especially of the RNA which is synthesized in the nucleoli and then passes into the cytoplasmic ribosomic RNA.

Treatment with Da on synchronized cells shows a marked drop of the mitotic index and appearance of mitotic anomalies when the cells are treated in S and G2 phases.

This reduction keeps also in the second mitotic wave. A reduction of the mitotic activity is observed also if the cells are treated near the middle of the interphase.

Da causes a reduction in the percentage of the nuclei labelled with 3H thymidine and in the number of grains per nucleus, when the cells are treated in S phase, in the very beginning and middle of interphase.

A report is given on various forms of administration of Trenimon for local treatment of neoplasms of the serous membranes. For supervision and adjustment of the chemotherapeutic effect supravital staining with acridin orange and phase-contrast microscopy of the untreated effusion sediment are especially suitable. This facilitates distinction between newly formed and preformed granules, which is of importance for the diagnosis. Inhibition of dye-induced production of granules is a very sensitive criterium for assessment of cell vitality. The development of regressive changes in the tumour cells caused by chemotherapy is discussed with reference to micrographs of a metastatic pleural sarcomatosis. Electron-microscope studies on the exudate suggest that prior to necrobiosis an increased transfer of the granular nucleolar component into the cytoplasm takes place. This may be caused by a true increase in the transfer of nucleolar RNA towards the cytoplasm or by an inhibition in the late S-phase.

1. Trenimon has three remarkable pharmacological characteristics, namely: very low toxicity, easy way of employing, big therapeutic action.

2. It results particularly useful in lymphatic and myeloid chronic leukemias, Vaquez's disease, hemorrhagic thrombocythemia, Waldenstrom's disease. It has to be noticed that Trenimon action is valuable in chronic myeloid leukemia even when it has been already treated with Busulphan for a long time.

3. Good results are obtained also in several cases of Hodgkin's disease, while the drug is less efficacious in Lympho-reticulosarcoma. Uncertain still being its action in plasmacytoma.

The authors report 13 observations concerning solid tumors treated with 5-fluoro-2-deoxyuridine.

11 of the patients have received protracted infusions at the rate of 1 mg/kg/24h during about a week.

2 patients have received the drug by direct intravenous injection at the rate of 15 mg/kg/24h during 6 days for one of them and during 12 days for the other one. These two methods are discussed according to the results and toxicity of both of them.

Natulan (Procarbazine) is a methylhydrazine derivative which in animal experiments inhibits a series of transplantable tumours. The Walker carcinoma is markedly affected. Even well established Walker tumours disappear completely under Natulan treatment. Tumour tissue is replaced by fibrous tissue. Natulan does not act selectively on neoplastic tissue, it also depresses the growth of other rapidly proliferating tissues such as the bone marrow.

As regards the mechanism of action the following data are known: Natulan lowers the mitotic index by prolonging the interphase and hindering the cell from entering into mitosis. In Ehrlich ascites tumour cells chromatid breaks were observed. Natulan depolymerizes deoxyribonucleic acid (DNA) in vitro. In vivo the synthesis of DNA is inhibited. Furthermore alkylation of purine bases has been demonstrated. All these mechanisms may be responsible for the cytotoxic effect of methylhydrazine derivatives. Natulan does not possess cross-resistance towards other cytotoxic agents. This phenomenon is a further proof of its particular mechanism of action.

The Authors analyse the action of MH on the chromosomes of normal myeloid cells. They evaluate the indications, therapeutic activity and the limits of this substance on Hodgkin disease.

Notre expérience clinique sur le Natulan est encore plutôt limitée. Depuis six mois seulement il nous a été possible obtenir ce médicament et nous n'avons traité jusqu'à présent que seize malades, tous par thérapie orale.

Dans le tableau suivant nous indiquons les maladies dont étaient atteints nos patients et le résultat du traitement.

Quelqu'une de nos observations mérite un bref commentaire.

Le premier malade que nous avons soumis au traitement était atteint de M. de Hodgkin généralisée, désormais insensible à toute thérapie connue. Le malade était en effet très près de la mort. Et bien, le Natulan eût une influence bénigne dramatique, soit pour la rapidité, soit pour l'entité de la rémission. Mais après un mois il y eût une rechute et, malgré la therapie d'entretien, le patient est mort en cachéxie. Dans ce cas nous n'avons pas observé de phénomenès toxiques sévères sauf une légère polynevrite des membres inférieurs.

Il faut encore remarquer que le Natulan a été actif aussi contre des hémoblastoses différentes de la M. de Hodgkin, c'est-à-dire le myélome, la M. de Waldenström et le lymphosarcome. Mais dans ces cas les rémissions ont été relativement plus rares et moins remarquables que dans la M. de Hodgkin.