[Interaction between immunomodulatory drugs in mice with special regard to changes of drug sensitivity and rate of bacterial translocation]. / Immunmoduláns anyagok kölcsönhatása egérkísérletekben különös tekintettel gyógyszerérzékenység változásra és a bakteriális transzlokáció alakulására.

In acute toxicity experiments the changes in drug sensitivity and in the rate of bacterial translocation (BT) were investigated in mice treated with immunomodulatory drugs: dianhydrogalactitol (DAG) in doses 20 and 30 mg/kg, chlorpromazine (CPZ) in doses 60 and 75 mg/kg and Mannozym (M) in dose 40 mg/kg for zymosan content. The drugs were used separately or in combination. The sensitivity of mice to immunosuppressive DAG or CPZ was higher in the case of combined treatment, than that of separately treated ones. The rate of BT was also higher in combined treated mice. The pretreatment with M that has immunostimulatory effect, influenced neither the sensitivity of mice to DAG or CPZ, nor the normal very low rate of BT. The present results reinforced the authors' earlier observations, that the effects of immunosuppressive drugs could cumulate and cause more serious damage of the organism. The authors suggest that the increase in drug sensitivity to immunosuppressive agents is in connection with increased rate of BT and effect of endotoxin.

Interaction of immunomodulant substances in mouse experiments with special regard to drug sensitivity and bacterial translocation.

In acute toxicity experiments changes in drug sensitivity and in the rate of bacterial translocation (BT) were investigated in mice treated with immunomodulatory drugs: dianhydrogalactitol (DAG) in doses 20 and 30 mg/kg, chlorpromazine (CPZ) in doses 60 and 75 mg/kg and Mannozym (M) in dose equivalent to 40 mg per kg zymosan. The drugs were used separately or in combination. The sensitivity of mice to immunosuppressive DAG or CPZ was higher in the case of combined treatment than that of separately treated ones. The rate of BT was also higher in mice receiving combined treatment. Pretreatment with M exerting an immunostimulatory effect, influenced neither the sensitivity of mice to DAG or CPZ nor the very low normal rate of BT. The present results reinforced the authors' earlier observations that the effects of immunosuppressive drugs cumulated in and caused more serious damage of the organism. The increase in drug sensitivity to immunosuppressive agents may be connected with an increased rate of BT and effect of endotoxin.

Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.

Loss in cell killing effectiveness of anticancer drugs in human gastric cancer clones due to recovery from potentially lethal damage in vitro.

The ability of human gastric cancer clones to recover from potentially lethal damage was studied. Recovery was greatest following treatments with bleomycin or Adriamycin; the recovery ratios (i.e., survival) increased almost 8-fold during a posttreatment incubation period. Recovery was also possible following treatments with actinomycin D, 1,2:5,6-dianhydrogalactitol, and diaziquone; however, the recovery ratios never increased above 2. No recovery was observed following treatment with 5-fluorouracil. Recovery from potentially lethal damage may be related to the heterogeneity in survival responses observed following treatment with some anticancer drugs. Bleomycin and Adriamycin treatments result in large heterogeneous survival fractions among these human stomach cancer clones, and the potentially lethal damage recovery ratios were larger (and variable). However, actinomycin D, diaziquone, and 1,2:5,6-dianhydrogalacticol produce very uniform killing effects in these cells and the recovery ratios are very much smaller and less variable. Finally the large amount of recovery observed after bleomycin or Adriamycin treatments resulted in the loss of cell killing effectiveness of the agents. Because the survival fractions increased during the recovery period, the net effect on cell killing was reduced to an amount normally obtained with doses that were up to six times smaller.

Relation between dose, plasma concentration and toxicity in a phase I trial using high dose intermittent administration of an alkylating agent, diacetyldianhydrogalactitol (DADAG).

Diacetyldianhydrogalactitol (DADAG), a new alkylating sugar alcohol derivative, was administered as single, 30-min infusions in doses ranging from 390 to 1200 mg/m2. The dose-limiting toxicity was myelosuppression. The median times to WBC nadir and regeneration were 16 and 21 days, and to platelet nadir and recovery 20 and 27, respectively. Nausea and vomiting occurred frequently and were of moderate severity. For phase II studies 900 mg/m2 DADAG given every 4-6 weeks is recommended. The area under the plasma concentration time curve (AUC) for DADAG did not increase in proportion with dose escalation; it changed only from 235.5 +/- 70.7 to 262.4 +/- 71.5 micrograms h ml-1 between doses of 690 and 1050 mg/m2. No correlations between the dose administered and the nadir values for haemoglobin concentration, WBC and platelet counts, or the number of episodes of vomiting were demonstrable in this dose range. Such an association was revealed, however, when the above biological variables were related to the individual AUC for DADAG.

Biochemical changes of intestinal epithelial cells induced by cytostatic agents in rats.

In the intestinal epithelium the rapidly proliferating crypt cells, the precursors of the mature enterocytes are extremely sensitive to the effects of cytostatic agents. The symptoms of intestinal impairment: nausea, vomiting, diarrhea, ulceration, are well known both in clinical practice and in experimental chemotherapy. To obtain information about the biochemical nature of these side effects, a study was performed by investigating the influence of clinically used alkylating hexitol derivatives, dianhydrogalactitol and diacetyl-dianhydrogalactitol, on rat intestinal mucosa cells. The biochemical parameters were investigated in isolated intestinal mucosa cells. Cell proliferation was characterized by measuring the activity of thymidine kinase, while digestion was evaluated by assaying the alkaline phosphatase, sucrase and maltase activities localized in the brush border membrane of the villus cells. The dose response studies of the different enzyme activities indicated that inhibition in all cases was dose dependent. The nadir of the intestinal damage and the time of regeneration were influenced both by the dose and the dosage schedule of the drugs.

Toxicity, antitumour and haematological effects of 1,2-anhydro-6-bromogalactitol and d-mannitol: a comparison with the related dibromo- and dianhydro-derivatives.

1,2-Anhydro-6-bromo-6-deoxygalactitol (BrEpG) and its D-mannitol analogue (BrEpM) intermediary metabolites in the conversion of dibromodulcitol (DBD) and dibromomannitol (DBM) into dianhydrogalactitol (DAG) and dianhydromannitol (DAM) have been prepared. The three types of derivative of each hexitol have been compared in their toxicities towards mice, tumour inhibitory activities against the Walker carcinosarcoma and haematological effects in rats. The bromoepoxides showed intermediate potency in all tests. The galactitol derivatives were always more potent than their mannitol counterparts. The mannitol derivatives were selectively myelosuppressive, being twice as toxic towards granulocytes as towards lymphocytes. The lymphotoxic activity of DBM, in particular, relative to its other toxic effects was particularly mild. These differences have been ascribed principally to the more rapid reactivity of DAG compared with DAM towards target nucleophiles, modulated by the influence of the bromine substituent on the transport properties of the dibromo- and bromoepoxy-derivatives.

Effect of phenobarbital on the toxicity and antitumor activity of dibromodulcitol and dianhydrogalactitol.

5-Ethyl-5-phenylhexahydropyrimidin-2,4,6-trione (phenobarbital) pretreatment significantly decreased the acute toxicity of 1,6-dibromo-1,6-dideoxygalactitol (dibromodulcitol) in H/Riop-Swiss mice and Wistar rats. Toxicity of dianhydrogalactitol, however, was not influenced to a considerable measure. Phenobarbital did not alter in itself the growth rate of the tumours examined. It significantly reduced, however, the antitumor activity of dibromodulcitol in NK/Ly-mouse lymphoma and Yoshida rat tumor. In contrast, the cytostatic effect of dianhydrogalactitol was not influenced at all by phenobarbital. The dissimilar effects of phenobarbital on the toxicity and anti-tumour activity of dibromodulcitol and dianhydrogalactitol are probably due to differences both in their metabolism and pharmacodynamics.

Mutagenicity of dibromodulcitol (DBD), an alkylating anticancer drug) and its mono- and bifunctional conversion products studied by the Salmonella/microsome assay.

Dibromodulcitol (DBD) and one of its most important bifunctional transformation products, dianhydrogalactitol (DAG) with similar cytostatic effect, were tested by the Salmonella/microsome assay on strains TA 1535, TA 1538, TA 98 and TA 100 using the plate incorporation technique. Both drugs were direct mutagens in strains TA 1535 and TA 100 and non-mutagenic in other strains. Their mutagenic effect was not influenced by S-9 mix from rat liver. Mutagenicity of DAG was very limited because of its marked toxicity. The other monofunctional alkylating derivatives, i.e., 1-bromo-3,6-anhydrodulcitol and 1,2-epoxy-3,6-anhydrodulcitol were highly mutagenic in strains TA 1535 and TA 100 with and without S-9 mix despite having no anticancer effect. Anticancer activity was exerted only by the bifunctional alkylating hexitols (DBD, DAG) which showed moderate mutagenic activity compared to the monofunctional derivatives. No correlation could be established between the mutagenic and anticancer effect of the four structurally related hexitols. Mutagenicity of urine and bile from rats after a single administration of the maximum tolerated (450 mg/kg) dose of DBD was also examined, and the hexitol components of the same urine sample were identified by t.l.c. DBD and its mutagenic transformation products were excreted in urine and not through the bile. The mutagenic effect of DBD observed cannot be attributed exclusively to DBD observed cannot be atributed exclusively to DBD itself, because the parent molecule, like other alkylating agents, easily undergoes spontaneous decomposition under in vitro and in vivo conditions to release both bi- and monofunctional alkylating solvolysis products and there highly reactive derivatives may play a role in this effect. No significant difference in the relative mutagenicity was detected between DBD and cyclophosphamide, used as a reference substance.

Higher resistance of germfree mice to dianhydrodulcitol, a lymphotropic cytostatic agent.

The same dose of dianhydrodulcitol (DAD) produced a lower mortality rate among germfree mice than among SPF or conventional C3H mice. On the other hand, it caused graver lymphoid atrophy in germfree mice. Their higher resistance, as evidenced by the mortality rate, can be explained on the basis of a histological study of the ileum. It showed milder alterations of the intestinal wall in germfree than in SPF mice. The lymphotropic cytostatic agent had a less direct toxic effect in germfree mice, due to the lacking damaging effect of endotoxin from the normal intestinal flora.

Dianhydrogalactitol and neural tumors: an in vitro, in vivo preclinical evaluation.

The effects of dianhydrogalactitol on human neuroblastomas in vitro and in vivo as heterotransplants in nude mice were determined. Four neuroblastoma lines and three primitive neuroectodermal tumor lines were found, in vitro, to have different sensitivities to the drug. The most sensitive in in vitro assays was the neuroblastoma line SK-N-Mc. Tumors from patients resistant to cyclophosphamide were sensitive to dianhydrogalactitol in vitro and in nude mice. The lack of increased cytotoxicity in vitro with concentrations greater than 12 micrograms/ml and the lowered degree of weight loss in mice treated with 6 mg/kg/day x 5 consecutive days compared to 15 mg/kg/day x 2 days (either in sequence or with a 3-day interval) suggest that clinical trials with 5-day courses or constant infusions may be more effective than intermittent pulsed doses.

Phase II trial of dianhydrogalactitol in metastatic malignant melanoma: a Southwest Oncology Group study.

Dianhydrogalactitol given iv in a schedule of 30 mg/m2/day for 5 consecutive days every 4 weeks was administered to 27 patients with metastatic malignant melanoma. All patients had received extensive prior therapy including chemotherapy and had progressive disease at the start of the study. Of 24 patients evaluable for response, 21 demonstrated progressive disease and three had stable disease for periods of from 4 to 11 months. No objective responses were observed. Two of the remaining three patients died 6 and 10 days after entry in the study, while the third refused to return after one drug course. Adverse effects included myelosuppression in eight patients, nausea and vomiting in five patients, and alopecia in one patient. Dianhydrogalactitol is considered to be insignificantly active in the secondary treatment of metastatic malignant melanoma at the dose and schedule studied.