Synthesis, structure and antitumor activity of new benz[d,e]isoquinoline-1,3-diones.

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.

Benzimidazo[1,2-c]quinazolines: a new class of antitumor compounds.

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.

Synthesis of new derivatives of flavone-8-acetic acid.

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.

Bis-naphthalimides: a new class of antitumor agents.

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.

In vivo administration of tumor necrosis factor-alpha is associated with antiviral activity in human peripheral mononuclear cells.

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.

Circumvention of multidrug resistance mediated by P-170 glycoprotein using calcium antagonists in primary human renal cell carcinoma.

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.

Effect of intraperitoneal recombinant human tumour necrosis factor alpha on malignant ascites.

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.

[Therapy of ascites with tumor necrosis factor in ovarian cancer]. / Aszites-Therapie mit Tumornekrosefaktor beim Ovarialkarzinom.

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.

Effects of calcium antagonists in multidrug resistant primary human renal cell carcinomas.

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.

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

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.

Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).

MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

[Role of calcium antagonists in the treatment of chemoresistant cancer of the kidney]. / Rôle des antagonistes de calcium dans le traitement des cancers chimiorésistants du rein.

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.

Tumour necrosis factor production and natural killer cell activity in peripheral blood during treatment with recombinant tumour necrosis factor.

Tumour necrosis factor (TNF) has been found to be an important immunomodulator. Among other functions TNF activates natural killer (NK) cells and stimulates monocytes/macrophages in an autocrine fashion. TNF production and NK activity in peripheral blood mononuclear cells were determined in a clinical phase I study in which recombinant human (rh) TNF was administered as a continuous infusion weekly for a period of 8 weeks. Even though TNF production and NK activity were significantly reduced directly after rhTNF infusion the effect proved to be transient and most pronounced at the first rhTNF administration. One day after completion of the rhTNF infusion the peripheral cells released more TNF into the supernatant compared to TNF activity immediately before the rhTNF infusion. This effect was conspicuous in non-stimulated cultures. After repeated rhTNF infusions both stimulated and non-stimulated TNF production of the peripheral blood mononuclear cells was increased. NK cell activity was also enhanced after repeated cycles of rhTNF administration as compared to early rhTNF treatment. Thus, repeated rhTNF infusions lead to a stimulatory effect on TNF production and NK activity of peripheral blood cells.

Phase-I trial of intravenous continuous infusion of tumor necrosis factor in advanced metastatic carcinomas.

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.

Phase I study of recombinant human tumor necrosis factor alpha in advanced malignant disease.

A phase I study with recombinant human tumor necrosis factor alpha (rhuTNF-alpha; Knoll AG, Ludwigshafen, FRG) in patients with advanced malignant disease was undertaken to evaluate drug toxicity (organ specificity, time course, predictability, reversibility, maximal tolerated dose), effectiveness, antigenicity and pharmacokinetics. TNF was administered as a test dose followed by daily i.v. infusions for 5 days, every 3 weeks (single i.v. infusion lasting 10 min, TNF dissolved in 50 ml 5% human albumin). Dosage was increased in groups of 3 or 4 patients from 0.04 mg/m2 to 0.28 mg/m2. A total of 19 patients with different cancers, including seven large-bowel carcinomas, three chronic myelogenous leukemias, three hypernephromas, two small-cell lung cancers, one malignant melanoma, one malignant lymphoma, one rhabdomyosarcoma and one fibrosarcoma were treated. Major side-effects were chills and fever (maximum 40.4 degrees C, median 38.7 degrees C, 19/19), headache (12/19), nausea and vomiting (12/19) and pronounced (greater than 20%) hypotension (4/19). Acute side-effects could be diminished by paracetamol or indomethacin pretreatment, and with one possible exception no tachyphylaxis to TNF was noted. Mild renal toxicity was seen during TNF treatment. Pharmacokinetic studies showed a serum half-life (t1/2) ranging from 11 min to 17 min for doses from 0.04 mg/m2 to 0.16 mg/m2 and prolonged clearance with t1/2 ranging from 54 min to 70 min in the 0.20-0.28 mg/m2 dose range. No objective antitumor effects were observed in this phase I study.

Decrease of natural killer cell activity and monokine production in peripheral blood of patients treated with recombinant tumor necrosis factor.

Tumor necrosis factor (TNF), a protein predominantly produced by activated macrophages/monocytes, is presently available in recombinant, purified form for clinical trials. Intensive studies in many laboratories have shown that besides the tumorcytotoxic effects, TNF acts on a large array of different cells and has potent immunomodulatory activities. In a clinical phase I study, some immunologic functional parameters of blood cells from patients who received 24-hour infusions of recombinant human TNF (rhTNF) were analyzed. Natural killer (NK) cell activity, TNF production, interleukin-1 (IL-1) production and mitogen-induced proliferation were measured either in whole blood samples or in cultures of peripheral mononuclear leukocytes of the patients directly before and after rhTNF infusion. NK cell activity, TNF and IL-1 production capacity and proliferative responses to concanavalin A (Con A) were significantly reduced after rhTNF application. We conclude from these observations that rhTNF in vivo acts directly or indirectly on NK cells and monocytes by either inactivating their functional capacity or by absorbing the relevant cells to the endothelial cell layer, thus removing them from circulation.

Immune response by biological response modifiers.

Several biological response modifiers (BRMs) were demonstrated to increase myelopoiesis and effector cell responses (M phi and natural killer cell activity) in vivo. The increased myelopoiesis was reflected by an increase in bone marrow cellularity and granulocyte-M phi colony-forming cells (GM-CFU-C). The increase in myelopoiesis appeared to be related to a concomitant increase in colony-stimulated factor (CSF) production and secretion by M phi and bone marrow cells. CSF induction by BRMs increased myelopoiesis and counteracted the myelosuppressive and immunosuppressive effects of cyclophosphamide. CSF induced in vivo by BRMs attained high titers and were maintained over a longer period than exogenously injected CSF, which was rapidly cleared from serum.

Protein deficiency reduces natural antitumor immunity.

Clinical data have shown that neoplastic diseases and/or related therapies frequently result in protein depletion of tumor-bearing patients. Depressions of acquired and specific immunity caused by protein depletion are well known. In an experimental model protein depletion was induced by lack of nutritional protein in otherwise isocaloric conditions in BALB/c and C57BL/6 mice over various time periods (max. 35 days). The results show that natural immune effector cells, natural killer cells, and monocyte/macrophages also during treatment with biological response modifiers (BRM) are depressed in their cytotoxic potentials in vitro and in vivo. Substantial and critical reductions of bone marrow cellularity (bone marrow nucleated cells) were also observed. In contrast, preliminary results show that if, following protein depletion, mice were treated parenterally with amino acids (Neo-aminomel, Boehringer-Ma. Co., FRG) complete restoration of immune parameters takes place. Adequate protein status is shown to be a crucial factor for natural immunity and therapy with BRM.