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New benz[d,e]isoquinoline-1,3-diones related to mitonafide (CAS 54824-17-8) and amonafide (CAS 69408-81-7) with double substitution on the chromophore and branched side chains have been synthesized and their biological activity determined. Molecular modeling studies of 3a based on X-ray crystallographic data of mitonafide have shown that the aromatic system intercalates between GC steps of DNA. The in vitro cytotoxic test data using CX-1 and LX-1 cells showed higher cytotoxic activities in disubstituted derivatives compared to both lead compounds. Some of the compounds have been selected for in vivo test using L1210 tumor cells and CX-1 cells. Two of them (3b and 3j) have shown promising activity as good candidates for clinical development.

A series of substituted benzimidazo[1,2-c]quinazolines have been synthesized. This class of compound has been designed by structural comparison with other intercalator patterns. The determination of in vitro activities has shown high inhibitory values.

The synthesis of new flavone-8-acetic acid derivatives is described. It is studied the influence of a dialkylaminomethyl group in the 3-position on the activity. None of the new compounds showed cytostatic activity.

A series of bis-intercalators bearing the 1,8-naphthalimide chromophore has been synthesized and in vitro activities determined. Most compounds have higher activities in HT-29 than the leader compounds Mitonafide and Amonafide. One of them (22) was selected for in vivo studies and presents an interesting activity in MX-1 and OVCAR models. (22) also acts as antitopoisomerase II.

Tumor necrosis factor-alpha (TNF-alpha) has a spectrum of biologic effects and has been shown to exert antiviral effects in fibroblasts in vitro. The in vivo administration of TNF-alpha (40-160 micrograms/m2 intravenously over 2 hr) and its effects on vesicular stomatitis virus (VSV) replication in peripheral blood mononuclear cells (PBMC) from patients with malignancy was investigated. Blood was obtained before, during, and after infusion. The PBMC were separated and infected with VSV at a multiplicity of infection of 0.005 plaque-forming units/cell and virus yields were determined 72 h later. The TNF-alpha inhibited VSV yields by as much as 99% in a dose-dependent manner with the inhibition initially observed during the first hour of infusion. Despite a rapid reduction in TNF-alpha serum levels, the higher doses still produced antiviral effects 4 hr after the infusion. Sera obtained at identical times had no interferon activity. Human gamma-interferon (25 micrograms/ml) added in vitro augmented the TNF-alpha-induced inhibitory activity in both magnitude and duration. Percentages of lymphocytes and monocytes in peripheral blood were reduced at 4 hr after TNF-alpha administration and the monocyte to lymphocyte ratio was diminished and temporally coincided with the loss of TNF-induced antiviral state. These data suggest that the in vivo administration of TNF has a direct inhibitory activity on VSV replication in human peripheral blood mononuclear cells that was enhanceable by gamma-interferon and possibly monocyte mediated.

In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance mediated by P-170 glycoprotein in vitro. So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded sufficient dosage. Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept. In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance. Concomitantly, P-170 glycoprotein expression was traced immunohistochemically using moab C 219. Substances derived from piperazine (flunarizine) showed only minor effects in this respect. The calcium antagonists of the papaverine type such as verapamil etc. revealed the strongest reversal of chemoresistance. Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the verapamil activity. All calcium antagonists lead to a significant enhancement of vinblastine cytotoxicity. An obvious link of P-170 glycoprotein to vinblastine chemoresistance was demonstrated. This particular resistance characteristic was detected in 19 of 27 resistant cases, but in none of the tumors displaying a chemoresponse. In particular, the new stereoisomer R-verapamil, which showed strong reversal of chemoresistance but which exerts 10 times lower cardiovascular side effects than racemic verapamil, seems to be suitable for further evaluation with regard to the clinical application.

29 patients with refractory malignant ascites due to metastatic peritoneal spread of adenocarcinomas originating from the ovary, gastrointestinal tract, liver, breast and uterus were treated in a phase I trial of intraperitoneal infusions of recombinant human tumour necrosis factor alpha (rhTNF-alpha). Patients received 40-350 micrograms/m2 rhTNF-alpha intraperitoneally once weekly for 2 months or for a shorter period in case of early resolution of ascites. Systemic side-effects resembled those reported for rhTNF-alpha given intravenously. No dose-limiting toxicities were found and thus a maximum tolerated dose of intraperitoneal rhTNF-alpha was not established. Out of 29 patients, 22 responded with a complete (16) or partial (6) resolution of their ascites. There was a less than 50% reduction in 4, and no increase in ascites in 1. 1 patient showed progressive ascites formation, and another patient was not eligible because of early death unrelated to treatment. Trials in patients with smaller tumour burden are warranted.

The biotechnological production of human recombinant tumour necrosis factor (rHuTNF) makes this drug available for clinical application. This endogenous compound exhibits tumouricidal activity and regulatory functions within the immune system. 20 out of 23 (87%) patients with refractory recurrent malignant ascites from ovarian cancer were successfully treated in a phase-I and II-study. The production of ascites was either completely suppressed or reduced to a minimum for at least 4 weeks after maximally three intraperitoneal (i.p.) applications. Two of the three non-responders were mucinous carcinomas. In the phase-I study the evaluation of a maximal tolerable dose was not possible due to the rapid therapeutic success at low doses of TNF. The effective dosage was 0.08-0.14 mg TNF/m2 given i.p. Side effects which occurred 2 to 24 hours after the application of TNF were flue-like symptoms combined with general malaise. The side effects were not dose related. All concomitant signs and symptoms could be minimized by prophylactic or therapeutic application of indometacine, paracetamol or pethidine. This applied especially for the typical early phase cytokine side effects e.g. chills and febrile temperatures. The side effects were not dose related. The i.p. treatment with rHuTNF appears to be a novel practicable and effective method for palliation in patients with recurrent ascites even in multiple pretreated patients.

Human renal cell carcinomas display a characteristically high degree of intrinsic chemoresistance to a multitude of chemotherapeutic agents. It was suggested previously, that P-170 glycoprotein contributes to this phenomenon in renal cell carcinoma indicated by elevated MDR-1 gene mRNA levels and by the expression of this specific resistance characteristic. The P-170-related efflux mechanism can be inactivated by certain calcium antagonists. P-170 was traced immunohistochemically using monoclonal antibody C 219. Concomitantly, we studied the enhancement of vinblastine cytotoxicity with 4 major classes of calcium-blocking agents in a microculture tetrazolium assay. Seven different calcium antagonists were selected: verapamil (VPM, racemic form), its R-stereoisomer (R-VPM), diltiazem, flunarizine, nifedipine, and its derivatives nimodipine and nitrendipine. Verapamil or R-verapamil causes a significant decrease of viable tumor cells as compared to vinblastine alone (P less than 0.001). Similar effects were found with diltiazem, nifedipine, and its derivatives reaching approximately 70% of the VPM/R-VPM activity. Flunarizine showed only minor enhancement of cytotoxicity. P-170 expression was demonstrated in 18 of 32 tumors, and a relation to chemoresistance was evident. None of the chemoresponders, but 18 of 25 (72%) of the highly resistant tumors, revealed this resistance factor. It was concluded that certain calcium antagonists in combination with chemotherapy may well offer therapeutic options in renal cell carcinoma as they apparently inactivate the underlying mechanism conferring resistance. The new stereoisomer R-VPM, in particular, may be used in clinical trials since it combines strong enhancement of vinblastine drug responsiveness with a 10-fold lower cardiovascular activity as compared to racemic VPM, thus allowing higher concentrations to be applied.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.

In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance in vitro. So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded a sufficient dosage. Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept. In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance. Substances derived from piperazine (flunarizine) showed only minor effects in this respect. The calcium antagonists of the papaverine type such as Verapamil etc. revealed the strongest reversal of chemo-resistance. Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the Verapamil activity. All calcium antagonists tested lead to significant enhancement of vinblastine cytotoxicity. In particular, the new stereoisomer R-Verapamil, which showed strong reversal of resistance but which exerts 10 times lower cardiovascular side effects than racemic Verapamil, seems to be suitable for further evaluation with regard to the clinical application.

Natural immuneffector cells, namely natural killer cells and monocyte/macrophages, have become in recent years an important part of theory and practice of immunotherapy. This is especially the case for therapy of malignant tumors with biological response modifiers (BRMs). Many BRMs increase cytotoxicity against tumor cells and cell numbers of natural immuneffector cells. Protein depletion on the other hand, which often is part of malignant diseases, results in well known negative effects on acquired and specific immunity. The data which are presented here show that depressions of natural immuneffector cells in terms of cytotoxicity and cell numbers also occur after protein depletion. The same is true for their pool, the bone marrow. The potentials of BRM therapy are considerably decreased. Host defense mechanisms in normal and protein depleted nutritional status, immunological tumor defense and BRM therapy are presented in the survey.

We have evaluated the possible role of biological response modifiers (BRMs) in myelopoiesis by investigating BRM modulated secretion of hematopoietic growth factors and inhibitors. Here, we report the evidence of augmented secretion of granulocyte and/or macrophage colony stimulating factors (CSF) by murine resident peritoneal macrophages after in vitro incubation with murine interferons (alpha, beta-mIFN; beta-mIFN; gamma-mIFN), poly ICLC (polyriboinosinic-polycytidylic acid poly-L-lysine), BM 41.332 (2-cyano-1-[(2-methoxy-6-methyl-pyridin-3yl)-methyl]-aziridine) and lipopolysaccharide (LPS). The secretion of CSF appears to be independent of the ability of the BRMs to induce IFN, as shown by the use of neutralizing antibodies against mIFN. The antiproliferative effects of IFN also did not block the BRM induced effects of CSF. The combination of alpha, beta-mIFN and poly ICLC or LPS and poly ICLC at suboptimal concentrations resulted in additive, but not synergistic effects on CSF secretion by macrophages. Histological examination of the colonies induced indicated the presence of two types of CSF, namely CSF1 and CSF3, which give rise to pure macrophage and granulocyte colonies respectively. In parallel to their effect on CSF secretion, these BRMs also caused a considerable increase in secretion of prostaglandins of the E series (PGE) by macrophages. However, the production of PGE did not interfere or influence CSF secretion, since the inhibition of the enzyme cyclooxygenase with indomethacin (10(-7) molar) 3 h before stimulation with poly ICLC, alpha, beta-mIFN, or LPS, inhibited the secretion of PGE by macrophages without affecting the secretion of CSF. Macrophages, stimulated by one of the active BRMs for 24 h, could not be restimulated by any of these agents to again secrete significant amounts of CSF or PGE, even after a 2 day resting phase. Other drugs tested (diethyldithiocarbamate, maleic anhydride divinyl ether, azimexone) failed to stimulate the in vitro secretion of significant amounts of CSF and PGE. The results presented here indicate that several BRMs can be utilized to stimulate macrophages to secrete the myelopoietic growth factor CSF, thus supporting the concept that these BRMs might be of value in reconstituting or promoting impaired granulocyte and monocyte/macrophage function.

We have compared the effects on number and function of bone marrow progenitors and peripheral effector cells of the myelomonocytic lineage of treatment with the 2-cyanaziridine compounds Azimexone and BM 41.332 to those of maleic anhydride divinyl ether copolymer (MVE-2) or granulocyte-macrophage colony stimulation factor (GM-CSF). Within a few hours after i.p. injection of either Azimexone or BM 41.332, there was a dose-dependent increase in serum CSF levels, CSF secretion by mononuclear bone marrow cells (BMC) and macrophages (M phi), which was followed by an increase in granulocyte-M phi committed stem cells (GM-CFU-C), nucleated BMC, and peripheral blood leukocytes. Optimal effects occurred 3 days after 50 mg/kg Azimexone or 25 mg/kg BM 41.332. Three i.p. injections of 50 mg/kg Azimexone into mice pretreated with cyclophosphamide (CY) (150 mg/kg) were able to significantly restore suppressed bone marrow cellularity (GM-CFU-C and nucleated BMC). Azimexone also increased the number of peripheral M phi in normal or CY-treated mice, without inducing detectable tumoricidal activity. These M phi, however, retained their capacity to become fully activated (cytotoxic) by appropriate activation signals such as IFN or LPS. Analogous to the 2-cyanaziridines. MVE-2 (at 25 mg/kg) had similar stimulatory effects on myeloid functions in normal mice. MVE-2 induced, in addition, a significant augmentation of cytoxicity by both M phi and NK cells. In contrast, single or multiple injections of semipurified GM-CSF into normal mice (1000 U or 5000 U per mouse) failed to detectably stimulate myelopoietic growth and differentiation. 2-cyanaziridine compounds thus offer the potential of selectively augmenting growth and differentiation of myelomonocytic cells in normal and bone marrow-depressed mice without appreciably affecting their immunological status.

Fifteen patients with advanced metastatic adenocarcinomas were treated in a phase-I study with continuous intravenous 24 h infusion of recombinant tumor necrosis factor alpha (TNF-alpha) in order to determine the maximum tolerated dose (MTD) and associated side-effects. Patients received 40-400 micrograms/m2 TNF-alpha once (arm A) or twice (arm B) weekly for a scheduled treatment period of 2 months. The observed systemic side-effects resembled those reported for interferons and included fever, chills, fatigue, headaches, myalgias, thrombocytopenia, prostration, and malaise. Dose-limiting toxicities, resulting in a median MTD of 200 micrograms/m2 for 24 h, were fever, chills, fatigue, myalgias, and thrombocytopenia. Out of 15 patients, 11 showed tumor progression, and 3 sustained in no change for over 2 months of treatment. A minor response was seen in 1 patient with a colorectal carcinoma and liver metastases. To reduce side-effects, patients were treated either with paracetamol or indomethacin. Higher MTDs were observed in patients treated with indomethacin. No detectable plasma TNF-alpha levels or TNF antibodies were measured under therapy (plasma TNF-alpha less than 20 pg/ml). We conclude that TNF-alpha appears to have some antineoplastic activity in patients with adenocarcinomas since 4 patients remained in no change or showed a minor response.