Uwe Bicker

In vivo antitumour activity of ilmofosine

Abstract Ilmofosine is a cytostatic/cytotoxic thioether phospholipid derivative. The in vivo anti- tumour activity of this compound was investigated in a methylcholanthrene (MethA)induced fibrosarcoma and in the 3 Lewis-lung carcinoma systems, respectively. Ilmofosine showed antineoplastic and antimetastatic properties at oral doses ranging from 0.625 to 40 mg/kg/day. Combination of Ilmofosine (p.o.) together with either cyclophosphamide (p.o.) or cis-DDP (i.v.) resulted in synergistic effects in vivo . These results demonstrate the in vivo antitumour activity of Ilmofosine in two tumour systems. The data indicate that direct cytostatic/cytotoxic effects of Ilmofosine are mainly responsible for its antitumour activity in vivo and which are increased by other cytotoxics.

Pharmacokinetics of the thioether phospholipid analogue BM 41.440 in rats.

BM 41.440 (1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine) is a cytotoxic thioether phospholipid analogue that recently has entered phase I trials in cancer patients. The objective of this study was to evaluate the pharmacokinetics of this compound in female rats after administration of a single oral dose (15 mg/kg body weight [bw]). Furthermore, BM 41.440 serum concentrations were determined under a daily oral treatment of up to 13 weeks. Blood samples were obtained via permanent catheters from the femoral arteries before and after drug administration for a total of 120 hr. Urine was collected in 24 hr-intervals for 120 hr; the volume was measured, and aliquots were stored at −20 C until analytical determination of the thioether derivative. BM 41.440 was assayed in serum and urine by means of a specific, newly developed reverse-phase high pressure liquid chromatography technique. Mean maximum serum concentrations (1.7 μg/ml, n=4 animals) were attained after seven hr. A terminal half-life of ca. 27 hr was calculated from the rate constant for the terminal elimination phase (λz ∼ 0.026/hr). The mean serum BM 41.440 concentration-time-area-under-the-curve was 52.9 mg × hr/l. The ratio of total body clearance to absorption fraction was 4.7 ml/min × kg bw. Only a small amount of the drug was found in the urine. The quantity excreted in the urine during a 24 hr-interval never exceeded 1.5% of the administered dose. Under a daily oral schedule (15 mg/kg bw × day) up to 13 weeks, mean BM 41.440 serum concentrations of 3.3±0.5 μg/ml and 5.2±1.2 μg/ml (mean ±S.D., n=10 animals) were found after five and 13 weeks, respectively. Taken together, the data indicate that BM 41.440 was absorbed from the gastrointestinal tract after oral administration and that accumulation of BM 41.440 can occur in rats.

Antineoplastic activity of the thioether lysophospholipid derivative BM 41.440 in vitro.

Thioether lysophospholipid derivatives (TLP) inhibited the in vitro uptake of [3H]thymidine into blasts of eight leukemias and cells of 12 different solid tumors of human origin. This effect correlated with trypan blue dye exclusion, which was used to assess cell damage.Cytostatic and cytotoxic effects of TLP were dependent on dosage and incubation time. Destruction of leukemic blasts was completed with >5 μg/ml after an incubation of >48 hr, but 10 to 20 μg/ml were necessary in solid tumors. Ester-linked 2-lysophosphatidylcholine was ineffective in the same dose range, which points to the requirement of the alkyl moiety insn-1 and a stablesn-2 substitution of the molecule for the antineoplastic effect.To assess putative antileukemic selectivity, the cytotoxicity (trypan blue dye exclusion) of TLP was compared in human cell samples of 19 non-neoplastic bone marrows and 9 leukemias. Results revealed a significantly higher activity of the TLP BM 41.440 in leukemic blasts.

Synthesis of thioether phosphocholine analogues

The synthesis of thioether phospholipids, which represent a new class of antitumor agents, is reported here. In particular, the route of synthesis of 3-hexadecylmercapto-2-methoxymethylpropyl-2′-trimethylammonio-ethyl phosphate (BM 41.440, Ilmofosine), one of the most potent cytostatic/cytotoxic derivatives, is described in detail. Starting with diethylbis-hydroxymethylmalonate, ethyl 2-phenyl-1,3-dioxane-5-carboxylate is formed via diethyl 2-phenyl-1,3-dioxane-5,5-dicarboxylate and 5-ethoxycarbonyl-2-phenyl-1,3-dioxane-5-carboxylic acid. Reduction of ethyl 2-phenyl-1,3-dioxane-5-carboxylate with LiAlH4 affords 5-hydroxymethyl-2-phenyl-1,3-dioxane. Alkylation with dimethyl sulfate gives 5-methoxymethyl-2-phenyl-1,3-dioxane. The ring structure then is opened byN-bromosuccinimide, resulting in the formation of 3-bromo-2-methoxymethylpropyl benzoate. Reaction of 3-bromo-2-methoxymethylpropyl benzoate with the sodium salt of hexadecanethiol leads to 3-hexadecylmercapto-2-methoxy-methylpropanol, which is reacted with a cyclic chlorophosphate to give the corresponding phosphorylated 3-hexadecylmercapto-2-methoxymethylpropanol. Treatment with trimethylamine yields BM 41.440. This compound already has been tested in clinical phase I/II trials in West Germany.

Animal experiments on the compensation of the immunosuppressive action of cyclophosphamide by 2-[-2-cyanaziridinyl-(1)-]-2-[-2-carbamoylaziridinyl-(l)]-propane BM 12 531

BM U2 531, the 2-[2-Cyanaziridinyl-(1)-]-2-[-2-carbamoylaziridinyl-(1)-]-propane, the further development of BM 06 002 is able to compensate the immunosuppressive action of Cyclophosphamide and to increase the carcinostatic action of Cyclophosphamide. These properties are demonstrated 1. by a leucocytosis induced after application of BM 12 531 in rats 2. by a quick restauration of leucocyte depression induced by Cyclophosphamide in rats and dogs 3. by an increase of resistance against an infection (candida albicans) in mice 4. by an increase of antitumour effect of Cyclophosphamide against a DS-carcinosarcoma in rats.

Suppression of Spontaneous Insulin-Dependent Diabetes in BB Rats by Administration of Ciamexone

BB rats spontaneously develop an insulin dependent diabetes which resembles in many features human type I diabetes. We have tested the effect of the immunomodulatory drug Ciamexone, a 2-cyan-aziridine-derivative, on the development of diabetes in BB rats. Ciamexone was given once daily during 6 days per week beginning with the age of 42 or 50 days up to 120 days. For comparison cyclosporin A (10 mg/kg) was applied following the same protocol. At 1 mg/kg ciamexone administration led to complete prevention of diabetes in females but was not beneficial in males. At 10 mg/kg the drug caused significant suppression of diabetes development in males but more pronounced in females. Both, a reduction of the incidence of diabetes and a delay in the onset of hyperglycaemia was observed only in females. After administration of cyclosporin A none of the animals developed diabetes. Ciamexone treatment did not affect granulocyte and lymphocyte counts and subsets in the peripheral blood except for a tendency to suppress eosinophilia. The growth of animals was not retarded. It is concluded that ciamexone seems to influence the autoimmune state of the BB rat resulting in partial suppression of the disease.

CIAMEXONE - A NEW HIGHLY SELECTIVE IMMUNOSUPPRESSIVE COMPOUND

A local graft versus host reaction between lymphocytes of Balb-c mice and its F-1-generation CB6F1 can be induced because of differences in products of the class II of the major histocompatibility complex (Ir). In this local mixed lymphocyte reaction mainly T-helper cells and B-cells are involved. An induction of a delayed type hypersensitivity reaction with oxazolon significantly decreases the local graft versus host reaction. Cyclosporin A depresses the local graft versus host reaction and the delayed type hypersensitivity in mice whereas Ciamexone, a 2-cyanaziridine derivative, very selectively only suppresses the local graft versus host reaction and increases the delayed type hypersensitivity.

Comparative Investigations of Various Immunoregulatory Substances in the Delayed Type Hypersensitivity Test of the Mouse

The substances D-penicillamine, auranofin, chloroquine, levamisole, BM 41.332, azimexone, bestatin, methisoprinol (inosiplex), thymosine (fraction 5), indomethacin and cyclophosphamide were examined comparatively in the delayed type hypersensitivity test after oxazolone sensitisation in mice. It was found, that only the basal antirheumatic drugs D-penicillamine, auranofin, chloroquine and levamisole and also BM 41.332 led to a potentiation of the DTH reactions. Methisoprinol, bestatin, azimexone, thymosine fraction 5 and indomethacin had no effect on the DTH, whilst the immunosuppressant cyclophosphamide led to an inhibition of the DTH reaction. It is concluded that this pharmacological model is suitable for screening of new basal drugs for rheumatoid arthritis.

Influence of BM 12.531 (Prop. INN azimexon) on the lymphocyte transformation and the percentage of active T lymphocytes in vivo and in vitro in man

SummaryPatients with different types of cancer received treatment PO or IV with the new immunostimulating compound, 2-[2-cyanaziridinyl-(1)]-2-[2-carbamoylaziridinyl-(1)]-propane, BM 12.531 (Prop. INN azimexon). After IV administration of 200 mg on 5 consecutive days or oral administration of 300 mg on 7 consecutive days a significant increase in the percentage of active T lymphocytes was seen. After in vitro incubation of different concentrations of BM 12.531 with blood from healthy donors there was a significant increase in the percentage of active T lymphocytes. No clear influence of BM 12.531 on the lymphocyte transformation caused by various concentrations of PHA could demonstrated because of the scatter.No side effects attributable to treatment with BM 12.531 were found, so that BM 12.531 is a good candidate for clinical trials.

Treatment of autochthonous rat colonic adenocarcinomas with a thioether-lysophospholipid derivative in mono-and combinationchemotherapy

SummaryIn 361 Sprague-Dawley rats autochthonous colorectal carcinomas were induced by intrarectal application of the carcinogen AMMN. Tumor-bearing animals were treated with a synthetic thiother-lysophospholipid (TLP) derivative and in combination chemotherapy with 5-fluorouracil (5-FU) and carmustine (BCNU). There was no difference in the survival time of treated and untreated animals. The median large-bowel tumor weight was significantly lower in the TLP/5-FU and TLP/5-FU/BCNU combination therapy groups than in the control groups. Transient hepatotoxicity was observed in the high-dosage (50 mg/kg body weight twice weekly) TLP group. This study confirmed the relative resistance of AMMN-induced colorectal carcinomas to antineoplastic treatment.