D-21266, a new heterocyclic alkylphospholipid with antitumour activity.

The aim of this study was to determine the antitumour effects of D-21266 in a rodent tumour model. Hexadecylphosphocholine (INN: Miltefosine) represents the first anticancer agent which was specifically formulated for topical use in cancer patients. The development as an oral drug was hampered by the gastrointestinal toxicity. Hexadecylphosphocholine derivatives were sought with a better therapeutic index. Octadecyl-(1,1-dimethyl-4-piperidylio) phosphate (D-21266) was identified as a suitable candidate. This compound is highly active in vitro inhibiting the growth of a number of human cancer cell lines. Mammary carcinomas were induced in Sprague-Dawley rats using DMBA, and oral doses of D-21266, in various schedules, were given to the animals. A high antineoplastic potency was observed without inducing loss of body weight at highly effective doses. The antitumour effect could be enhanced by introducing a dose schedule consisting of a high loading dose followed by a low maintenance dose, both of which are only marginally active when given alone. Therefore, D-21266 with its favourable pharmacological and toxicological profile, warrants evaluation in the clinic. However, the concept of clinical trials requires new approaches to dose finding and response evaluation, because the dose-response relationship of this compound is distinctly different from that of classical cytostatic agents.

Cellular signalling as a target in cancer chemotherapy. Phospholipid analogues as inhibitors of mitogenic signal transduction.

Mitogenic signalling mechanisms emerged as novel targets for tumor chemotherapy. Current strategies for pharmacological interventions are briefly discussed. Phospholipid analogues are treated in greater detail. It is shown here that this new class of antitumor agents acts as inhibitors of mitogenic signal transduction. The common target of all phospholipid analogues studied so far is the phosphatidylinositol (PI)-specific phospholipase C (PLC). This results in an attenuated formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The reduction in IP3-levels leads to a depressed release of Ca2+ from internal stores, and the reduced formation of DAG interferes with the growth factor-induced activation of protein-kinase C (PKC). In addition to the effect on PI-specific PLC, most phospholipid analogues inhibit PKC directly by interacting with the regulatory domain of the enzyme. This effect, however, is not observed with all phospholipid analogues. Some potent growth inhibitory representatives from this group like hexadecylphosphoserine or hexadecylphosphonoserine do not affect PKC in cell-free extracts. It is concluded, therefore, that the direct inhibition of PKC is not required for the growth-inhibitory activity of these agents. The ability of phospholipid analogues to interact with PKC was also not found to be correlated the occurrence of unwanted side effects. Phospholipid analogues have also been found to act as inhibitors of phospholipase D (PLD). However, in this case the correlation to the growth inhibitory potency of various phospholipid analogues was less clear, so that the contribution of the PLD inhibition to the growth inhibitory effect of these agents still remains to be established. The inhibition of the thrombin-induced rise in cytosolic free Ca2+ by phospholipid analogues is reversible by washing the cells in phospholipid-free medium. These findings suggest that phospholipid analogues do not cause persistent membrane damage and may act as cytostatic rather than cytotoxic agents.

Interference of new alkylphospholipid analogues with mitogenic signal transduction.

The interference of several new hexadecylphosphocholine analogues with mitogenic signal transduction was investigated in NIH3T3 fibroblasts by studying the effects of these agents on thrombin-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation and the subsequent Ca2+ release, on protein kinase C (PKC) in cell-free extracts, on the PKC-mediated activation of the Na+/H+ antiporter and on c-fos induction. The compounds investigated include hexadecylphosphocholine (HePC), octadecyl-[2-(N-methyl-piperidinio)-ethyl]-phosphate (D20133), octadecyl-(N,N-dimethyl-piperidinio-4-yl)-phosphate (D21266); octadecyl-[2-(trimethyl-arsonio)-ethyl]-phosphate (D21805) and hexadecylphospho-L-serine (HePS). The data indicate that (i) all compounds inhibit the thrombin-induced progression of growth-arrested NIH3T3 cells into S phase with similar IC50 values; (ii) the common denominator of all compounds is a reduction of Ins(1,4,5)P3 formation, resulting in an attenuation of Ca2+ release; (iii) the direct interaction with PKC does not significantly contribute to the antitumor activity of these agents; (iv) the new HePC congeners D21266, D21133 and D21805 affect the same targets as HePC, i.e. PKC and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC). The lower toxicities of these compounds cannot be explained by a less pronounced inhibition of PKC or PLC, respectively.

Opposite effect of miltefosine on the antineoplastic activity and haematological toxicity of cyclophosphamide.

The effect of pretreatment with miltefosine (MIL) on the antineoplastic activity of cyclophosphamide (CPA) was evaluated in subcutaneous benzo(a)pyrene-induced sarcomas (BPS) of the rat. MIL alone had no antineoplastic effect on this autochthonous tumour, but enhanced the chemotherapeutic effect of CPA. Conversely, MIL counteracted the myelotoxicity of CPA in normal adult rats. Although the nadir of the leucocyte count remained unchanged, the recovery phase was considerably shortened, an effect which resembled the pharmacological action of GM-CSF.

Determination of trace amounts of selenium in poultry feedstuffs by gas chromatography.

In view of the importance of establishing reliable selenium concentration levels in different kinds of feedstuffs, the purpose of this work was to develop optimum experimental conditions for the isolation and GC determination of selenium as its chelate with 4-nitro-1,2-diaminobenzene. It was shown that ignition of the sample in an oxygen flask followed by reduction of Se(VI) to Se(IV) and the formation of 5-nitro-2,1,3-benzoselenadiazole chelate in HCl medium is a relatively rapid procedure giving a low blank value and allowing the determination of selenium in commercial feedstuffs and similar biological samples. The method was validated by the analysis of suitable certified or standard reference materials.

Antineoplastic activity and tolerability of a novel heterocyclic alkylphospholipid, D-20133.

Octadecyl-[2-(N-methylpiperidinio)ethyl]-phosphate (OMPEP, D-20133), a heterocyclic analogue of hexadecylphosphocholine (MIL), has been synthesized in an attempt to increase the therapeutic range of the parent compound. The antineoplastic activity of the novel alkylphospholipid was compared with that of MIL in dimethylbenz(a)anthracene-induced mammary carcinoma of the rat. Using tumors of different sizes and repeated daily doses as well as high single doses, we achieved marked remissions with either compound. However, the therapeutic range of OMPEP was broader than that of the parent drug. Furthermore, the emetic potential of OMPEP tested on ferrets was distinctly less pronounced than that of MIL. In vitro the new alkylphospholipid proved to be more active than MIL in all cell lines tested, and its differentiation-inducing capacity turned out to be superior to that of MIL. No hematological toxicity was observed at various OMPEP doses during a 3-week treatment period.

Selective cytostatic activity of hexadecylphosphocholine against tumor-cells invitro leads to the establishment of an invivo screening system for phospholipid analogs.

Hexadecylphosphocholine (HPC, D-18506, INN: Miltefosine) was characterized in a clonogenic micro assay in vitro with respect to its cytotoxicity against a panel of human and murine tumor cell lines and murine bone marrow cultures (GM-CFC). The KB human epithelial carcinoma line was found to be more sensitive by at least one order of magnitude than the murine tumor cell lines which are in vivo insensitive to the drug. Xenotransplants of the KB cell line into nude mice were highly sensitive to HPC with optimal treatment resulting in total regression of the tumor. Twenty-seven structural analogues of HPC were tested in vivo in our normal screening system, the dimethylbenz(a)anthracene(DMBA)-induced mammary carcinoma of the rat, and against KB-xenotransplants. 88 % of the compounds rated identically in both models, no compound was classified falsely negative in the KB model. These results allow to adopt the KB-model, which is easier to perform, as a reliable primary screening system for phospholipid analogues without the risk of missing active compounds.

In vitro and in vivo antitumoral activity of alkylphosphonates.

Hexadecylphosphocholine is a new antitumour agent with a highly selective activity in chemically induced mammary tumours. It was suggested, that hexadecylphosphocholine is a pro-drug, cleavable by phospholipases C and/or D, creating hexadecanol or hexadecylphosphate as the active principle. To test this hypothesis, the antineoplastic activity of three alkylphosphonates, cleavable either by phospholipase C or D, are compared with those of the parent compound, hexadecylphosphocholine. Cell culture experiments, in which radiolabelled alkylphosphonates were incubated with a neoplastic cell line, showed no metabolism even after 3 days of incubation. In in vivo experiments with dimethylbenzanthracene-induced rat mammary carcinomas, all three alkylphosphonates showed antineoplastic activity, although none of them reached the high activity of hexadecylphosphocholine. These results indicate that the antitumoral activity of alkylphosphocholines and alkyl lysophosphatidylcholines is due to direct toxicity and not dependent on metabolism by phospholipases C or D or related enzymes.

Protection from ifosfamide-induced alopecia by topical thiols in young rats.

An animal model for testing substances that might prevent alopecia induced by oxazaphosphorines has been developed. It is based on the fact that the rat pups experience virtually total hair loss following administration of oxazaphosphorines. Using this model, we showed that epicutaneous treatment with sodium thioglycollate prevented ifosfamide-induced hair loss on the treated area. These experiments indicate that prevention of oxazaphosphorine-induced alopecia in man may be achieved by topical thiols.

Chloroacetaldehyde and its contribution to urotoxicity during treatment with cyclophosphamide or ifosfamide. An experimental study/short communication.

Based on clinical data, indicating that chloroacetaldehyde (CAA) is an important metabolite of oxazaphosphorine cytostatics, an experimental study was carried out in order to elucidate the role of CAA in the development of hemorrhagic cystitis. The data demonstrate that CAA after i.v. administration does not contribute to bladder damage. When instilled directly into the bladder, CAA exerts urotoxic effects, it is, however, susceptible to detoxification with mesna.

Experimental investigations into the carcinogenic effect of antitumor and immunosuppressive agents.

In a comprehensive experimental study on rats, the carcinogenicity of various therapeutically important antitumor drugs was investigated. The influences of strain, sex, dose and time of administration were systematically varied. The tested compounds showed remarkable differences in their carcinogenic potential. These differences were particularly obvious, not only when the total tumor rate was analyzed, but also when the distribution of tumor rates to the various localizations was considered (tumor spectrum). Three classes of carcinogenic substances were identified: (1) Substances showing a specific carcinogenicity: they lead to tumorogenesis primarily in organs or organ systems that, in the untreated control animals, remain virtually tumor-free for life. One example of such a substance is chlormethine. (2) Substances with non-specific carcinogenicity: they lead to an increase in tumors in organs that are also stricken in the control animals, however, to a clearly reduced extent. Oxazaphosphorine carcinogenicity is typical for this class. (3) Substances of mixed-type carcinogenicity: this group shows non-specific carcinogenicity, as well as a carcinogenic action with marked organ specificity. One example of this class is procarbazine. The antimetabolites tested were shown to be practically non-carcinogenic. Characteristic differences occurred between the two rat strains used in the investigation, Sprague-Dawley and BD II, with regard to the spontaneous tumor spectrum and the organ-related extent of carcinogenicity under the influence of the substances tested. In an experiment involving short-term application (up to 17% LD50, five times i.v. at 14-day intervals), the carcinogenic effects were substantially lower than in an experiment involving long-term application (up to 7% LD50, once a week for 52 weeks, i.v.), although the strain- and substance-specific characteristics in both experiments were rather similar.

Characterization of the antitumor activity of hexadecylphosphocholine (D 18506).

Hexadecylphosphocholine (HPC) differs from ether lipids with known antitumor activity by its lack of the glycerol part. In the experiments described here HPC revealed outstanding antitumor activity in dimethylbenzanthracene (DMBA)-induced rat mammary tumors. A dose-response relationship was seen after daily oral treatment with complete suppression of tumor growth at doses of 46.4 mg/kg/day. There was no schedule dependence and the therapeutic efficacy was independent of the tumor weight at the initiation of therapy. Another autochthonous tumor, the benzo[a]pyrene-induced sarcoma of the rat did not respond to HPC treatment, indicating a highly selective spectrum of activity of the test compound. In comparison to an optimal single dose of cyclophosphamide, a single high dose of HPC was considerably more active against the DMBA tumor. At therapeutic dose levels no major toxicity of HPC was observed. Bone marrow suppression was not encountered, on the contrary, at high doses leukocytosis became apparent. The available pharmacological and toxicological data suggest that HPC may be useful in the treatment of human cancer.

Pharmacokinetics and metabolism of sodium 2-mercaptoethanesulfonate in the rat.

The synthetic low-molecular-weight thiol, 2-mercaptoethanesulfonate (mesna), exerts efficient protection against oxazaphosphorine-induced urothelial toxicity by binding the renally excreted and concentrated toxic metabolite(s). In this study, the pharmacokinetics and metabolism of mesna and its disulfide form (dimesna) have been investigated in the intact rat and in several in vitro systems, including isolated perfused organs, freshly isolated cells, and subcellular fractions; the mechanism of reduction of dimesna to form the pharmacologically active thiol mesna has been further studied with purified enzyme preparations. The results may be summarized as follows: (a) After p.o. administration, mesna and dimesna are both absorbed from the intestine, and dimesna undergoes reduction to mesna during intestinal absorption; (b) when present in plasma, mesna is rapidly oxidized to dimesna by a metal-dependent reaction; (c) mesna and dimesna pass unchanged through the hepatic vasculature, are not taken up into liver cells, and are not excreted in bile; (d) in the kidney, dimesna is filtered through the glomeruli and subsequently reabsorbed, whereupon reduction to the pharmacologically active thiol form occurs in the renal tubular epithelium, and the thiol is then reexcreted into the tubular lumen; (e) reduction of dimesna to mesna occurs in intestinal and renal epithelial cells by a mechanism involving the cytosolic enzymes thiol transferase and glutathione reductase. Thus, the formation of the pharmacologically active thiol form from dimesna is associated with the consumption of equimolar concentrations of reduced glutathione.

Studies on the urotoxicity of oxazaphosphorine cytostatics and its prevention--III. Profile of action of sodium 2-mercaptoethane sulfonate (mesna).

Mesna is a pharmacologically unremarkable, physiologically largely inert and almost totally non-toxic thio compound. It is rapidly eliminated renally and only slightly permeates into tissues. It has been shown experimentally that the bladder damage inducible in the rat by administration of oxazaphosphorine cytostatics can be successfully prevented by quite small doses of mesna. The detoxifying action of mesna is limited to the kidneys and the efferent urinary tract. The systemic effects of the oxazaphosphorines, however, remain unaffected. This applies particularly to the curative oncocidal efficacy of these compounds. It has also been shown experimentally that mesna does not affect the curative effects of other cytostatic drugs (doxorubicin, BCNU, methotrexate, vincristine). The efficacy of the cardiac glycoside proscillaridin is also not impaired by mesna.

[Prevention of urotoxic actions of cyclophosphamide and ifosfamide by dimesna (preliminary communication) (author's transl)]. / Verhütung urotoxischer Wirkungen von Cyclophosphamid und Ifosfamid durch Dimesna (Vorläufige Mitteilung).

Sodium 2-mercaptoethane sulfonate (mesna, Uromitexan) is oxidized in the organism of rats to 2,2'-dithiodi-(ethane sodium sulfonate) (dimesna). Dimesna is partially reduced to the mercapto compound mesna (kidneys); both compounds are eliminated via the urine. Even after administration of dimesna mesna can be detected in the urine. Accordingly dimesna also proved to be an effective antidote against the urotoxic actions of cyclophosphamide and ifosfamide.