The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. III. Human serum albumin-methotrexate derivative, its preparation and basic testing.

The preparation and a more detailed characterization of human serum albumin-methotrexate derivative (HSA-MTX) is described. The synthesis of the derivative was performed by means of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (in methoiodide form) (WSC). Results of many experiments showed that, on the average, about 26 molecules of methotrexate (MTX) were coupled to one molecule of human serum albumin (HSA). The relative molecular weight of the formed derivative was estimated by gel chromatography on Sepharose 6B or on high-pressure liquid chromatography (HPLC) on SWK column, respectively. From the obtained data it follows that a considerable part of the HSA-MTX derivative formed protein-protein conjugate (up to about 4 X Mr HSA), nevertheless the derivative retains its good solubility as a native albumin. In order to eliminate the possibility of influencing the cytostatic activity of the derivative with byproducts of its synthesis, human serum albumin-folic acid derivative (HSA-FA) was prepared and tested by the same method. All demonstrated experiments proved that MTX was the only compound possessing the cytostatic activity. During the experimental therapy of Gardner lymphosarcoma (LSG) the following was found: (1) The intratumorous application of the drug was the most effective way of administration. (2) Any type of administration of the HSA-MTX derivative exerted a better effect than the same way of administration of free MTX. (3) The comparison of two (repeated) administrations of both drugs showed clearly that the HSA-MTX derivative was more efficient than free MTX. After HSA-MTX derivative treatment all animals survived without tumor. (4) For the estimation of the toxicity of the HSA-MTX derivative, three times and five times repeated intraperitoneal administration was performed. It was concluded that although the derivative was more toxic than free MTX, its therapeutic activity was better. After the elimination of the toxic manifestation of the HSA-MTX derivative by a suitable arrangement of drug doses, five times higher efficacy of the derivative was reached, as compared with free MTX. (5) The therapy by the HSA-FA derivative did not exhibit any therapeutic effect. The reason why HSA was used as a macromolecular carrier for cytostatics is discussed.

Organ distribution of fibrinogen-bound methotrexate in mice with Gardner lymphosarcoma.

The distribution of fibrinogen-bound 3H methotrexate was investigated in Gardner lymphosarcoma bearing mice. 3H labeled methotrexate (3H MTX) was covalently bound by means of aminopropyl carbodiimide to bovine and mouse fibrinogen (FBG). The preparations as well as the free 3H MTX were applied i.v. in a single dose to three groups of C3H mice on day 6 after the inoculation of Gardner lymphosarcoma. 3H MTX level was determined in the blood, spleen, tumor and liver. Sufficient amounts of MTX were released by proteolysis of FBG-MTX derivatives to induce chemotherapeutical effects. Protracted accumulation of MTX applied in the form of FBG-MTX derivatives was found in the spleen and in the liver, in contradistinction to free drug application, suggesting the proteolytic degradation as a directing step responsible for the prolonged persistence of FBG-MTX derivatives in the organs. In the tumor the highest amount of MTX was released from mouse FBG supporting the view of ready uptake of homologous FBG by tumors.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. II. Chemotherapy of Gardner lymphosarcoma with pea seed lectin-methotrexate derivative.

It was demonstrated that the tumorigenicity of tumor cells preincubated in low concentration of free methotrexate (MTX) has not been changed. On the other hand the preincubation of these cells with pea seed lectin (PL), MTX and PL mixture and especially pea seed lectin-methotrexate derivative (PL-MTX) influenced markedly the tumor cells tumorigenicity. The chemotherapy of Gardner lymphosarcoma (LSG) bearing mice with PL-MTX derivative was performed. After one dose therapy of LSG ascitic form with PL alone no effect on mice survival time was observed. The administration of PL-MTX derivative was efficient at a higher dose only. But free MTX was effective at both examined doses. Four times repeated injection of lectin to mice bearing the ascitic form of LSG shortened the survival time of mice. Repeated application of the higher dose of free MTX was accompanied with a considerable number of toxic deaths, but the life span of most surviving animals was prolonged. The similar but less expressive result has been reached by using PL-MTX derivative. The post mortem examinations suggest that the marked local inflammatory reactions are probably caused by the PL cytotoxicity. Therapy of solid LSG bearing mice with four i.t. injections of PL had no obvious effect on the survival. PL-MTX derivative prolonged distinctly the life span of tumored mice but the administration of sole MTX was the best.

Immunogenicity of the Gardner lymphosarcoma for the mice of the strain C3H (H-2k). II. Alteration of the growth and dissemination of solid Gardner lymphosarcoma following immunization by an extract of the ascitic tumor.

Aqueous 1-butanol extracts were prepared from the ascites form of Gardner lymphosarcoma (LSG) maintained in C3H (H-2k) mice and from the solid Lewis lung carcinoma (LLCa) transplanted in B6 mice. C3H mice primed at least two times with cell surface antigens extracted from Gardner tumor cells (LSG-extract) lived longer than untreated controls if challenged with the solid Gardner lymphosarcoma cells. Tumors growing in immunized C3H mice differed from those growing in controls by shape, necrotization and inhibition of dissemination. If C3H mice were primed three times with LLCa extract then the survival was in comparison to intact controls prolonged only when the mice were pretreated into the site of tumor challenge. Survival time of B6 and B10 mice with transplanted LLCa was not markedly changed by previous priming injections of LSG-extract. An increase in mortality was recorded when LSG-extract-primed B10 mice were compared to the group of mice of the same line; similar effect has not been found in B6 mice.

Immunogenicity of the Gardner lymphosarcoma for the mice of the strain C3H (H-2k). I. The effect of the 60Co-irradiation of recipients and of the interval between the immunization and transplantation of the tumor on the antitumor resistance of immunized mice.

The C3H (H-2k) mice were immunized by 60Co-irradiation-inactivated Gardner lymphosarcoma (LSG) cells. The degree of resistance of mice with transplanted tumors was determined by difference in survival curves of the immunized and nonimmunized mice. When the tumor was transplanted during 26 weeks after the last of the three immunizing injections the mean survival time of immunized mice was always prolonged over that of nonpretreated controls. The prolongation was not always significant. The slope differences between lines characterizing survival of immunized and nonimmunized groups were statistically significant when the transplantation of the tumor was performed up to the 22nd week following the immunization. A small number of immunized mice which survived 60 days without visible tumors belonged to immunized groups transplanted with the tumor up to 14 weeks after the last immunizing dose. A higher degree of resistance has been achieved in mice given increased number of immunizing injections. Similar effect was observed in mice with impaired antitumor resistance due to immunosuppressive dose of 60Co-irradiation given before the tumor transplantation. Skin grafts taken from resistant mice healed up to unaffected isologous mice as well as it was found in reciprocal transplantation experiments.

Treatment of C3H mice bearing solid Gardner lymphosarcoma with vaccine prepared from Corynebacterium parvum combined with methotrexate and Alexan.

C3H mice after transplantation of solid Gardner lymphosarcoma were injected with Methotrexate (MTX) or Alexan. The prolonged survival time of mice treated with antimetabolites was in some cases evidently reduced after an additional injection of Corynebacterium parvum (C.p.). This reduction did not depend on the sequence of corynebacterial vaccine or MTX injections. Significantly better effect of combined treatment with MTX injection and C.p. was found only once in mice preimmunized with the tumor irradiated from 60Co gamma source.

Effect of vaccine prepared from Corynebacterium parvum on the growth of Gardner lymphosarcoma in C3H strain mice.

The present paper discusses the effect of a vaccine prepared with formol-killed Corynebacterium parvum (alias Propionibacterium acnes) on the growth of solid Gardner lymphosarcoma transplanted on C3H (H-2k) mice. The vaccine was injected at various time intervals prior to, simultaneously with, or following transplantation of the tumor to intact mice or mice immunized with 60Co-irradiated Gardner tumor cells. The effectivity was evaluated in terms of numbers of mice surviving without tumor 60-day observation period or prolongation of survival time. Reliable effectivity showed vaccine (0.8 mg dry weight of C.p.) in the mixture with tumor transplant or when introduced 6 days earlier into the site of tumor transplantation. The preventive action of the vaccine was more pronounced in the mice immunized with inactivated tumor. The vaccine applied to mice with a transplanted tumor was ineffective and failed to significantly prolong the survival time even in mice which had previously been immunized with an inactivated tumor.

Effect of free and bovine fibrinogen-bound methotrexate and dibromoaminopterin on the dissemination of solid Gardner lymphosarcoma.

Solid Gardner lymphosarcoma transplanted subcutaneously to mice of strain C3H/Sumice does not grow solely at the site of inoculation, but disseminates also to remote tissues. An i. v. administration of fibrinogen-Methotrexate derivative or Methotrexate (8-15 mg antimetabolite per 1 kg) to mice with an early tumor, reduced the weight of the local tumor, prolonged their survival time and also the posttransplantation period during which the dissemination of malignant cells could not be detected. When the above substances were given to mice with advanced tumors whose dissemination could be reliably established, Methotrexate proved more effective than its fibrinogen derivative. 3.5'-dibromoaminopterin administered alone or bound to fibrinogen (14.6 mg of antifolate per kg) showed no effect both on survival rate and on tumor dissemination.

The chemotherapy of C3H mice bearing Gardner lymphosarcoma with bovine fibrinogen-methotrexate derivative.

Derivative of bovine fibrinogen (FBG) containing chemically bound methotrexate (MTX) has been prepared by action of ethyldimethylaminopropyl carbodiimide. The derivative retained its solubility and clotability. Intraperitoneally administered FBG-MTX derivative one day after transplantation of the ascitic Gardner lymphosarcoma prolonged distinctly the survival of C3H mice. The intravenous application of FBG-MTX derivative to mice bearing the solid form of the tumor exerted chemotherapeutic effect resulting in prolongation of survival. The local application of FBG-MTX solutions followed by injection of thrombin resulted in the formation of a fibrin clot in the tumor area which persisted at least 48 hours. Local chemotherapy of the solid tumor with fibrin clot containing MTX performed on day 1 or 3 led to significant prolongation of survival of the treated animals. Mechanism of MTX liberation and the possible application of FBG-MTX derivative in chemotherapy of tumors are discussed.

Changes in hemopoiesis of mice of the C3H strain following transplantation of Gardner lymphosarcoma and infection with LDH-virus. II. Spleen and bone marrow.

Cytological examination of spleens of mice with growing Gardner lymphosarcoma contaminated with LDH virus indicates the augmentation of erythropoiesis and granulopoiesis, as well as the decrease of lymphopoiesis. Similar changes were observed after infection with LDH-virus. The counts from the bone marrow smear showed more pronounced changes only in tumorous mice, in which repeatedly increased granulopoiesis was found. When the mice bearing Gardner lymphosarcoma were treated with L-asparaginase, then the cytological findings in the spleens and marrows of femors were almost normal. The examination of spleen and bone marrow smears revealed no tumor cells. Histologically examined spleens of mice with 12-day-growing Gardner lymphosarcoma demonstrated infiltration of the follicles with tumor cells. In the marrow of sternum no infiltration of tumor cells could be established.