Martin R. Berger

BCR‐ABL influences the antileukaemic efficacy of alkylphosphocholines

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr‐abl rearrangement (K‐562, LAMA‐84, CML‐T1 and BV‐173) and two other leukaemic cell lines (HL‐60 and SKW‐3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR‐ABL‐positive cell lines than the two reference APC. It induced programmed cell death in HL‐60 and SKW‐3 cells after exposure for 24 h, and in bcr‐abl expressing cells after a prolonged incubation period (48 h). LAMA‐84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast‐like phenotype. Experiments with a cell‐free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT‐PCR we confirmed that K‐562 and LAMA‐84 cells carry the b3a2 fusion type, and CML‐T1 and BV‐173 the b2a2 variant. BV‐173 cells had the lowest level of bcr‐abl mRNA which correlated with their increased sensitivity. Transfection of K‐562 cells with antisense oligonucleotides directed against bcr‐abl caused a specific suppression of K‐562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR‐ABL‐positive blasts as compared with miltefosine and perifosine. The expression of BCR‐ABL cannot prevent apoptosis but delays erucylphosphocholine‐induced programmed cell death. Transfection with bcr‐abl directed antisense oligonucleotides reduces the clonogenicity of K‐562 cells.

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.

Cilengitide inhibits progression of experimental breast cancer bone metastases as imaged noninvasively using VCT, MRI and DCE-MRI in a longitudinal in vivo study

The aim of this study was to investigate the effect of inhibiting αvβ3/αvβ5 integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ3 and αvβ5 integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle‐treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast‐enhanced (DCE‐) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle‐treated controls. DCE‐MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide‐treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ3/αvβ5 inhibition may be a promising therapeutic approach for bone metastases.

Distribution and metabolism of hexadecylphosphocholine in mice

Distribution and metabolic fate of radiolabeled hexadecylphosphocholine (He-PC) has been studied in mice. It is demonstrated that He-PC is well-absorbed from the intestinal tract, intravenous (IV) and oral administration lead to similar distributions throughout the body, the highest accumulation of radioactivity occurs in liver, lung and kidney, and the metabolic products are radioactive choline, phosphocholine and 1,2-diacylphosphatidylcholine. The occurrence of these metabolites indicates that phospholipases C and D may be involved in He-PC breakdown.

Osteopontin influences the invasiveness of pancreatic cancer cells and is increased in neoplastic and inflammatory conditions

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less then 5%. Invasive tumour growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis. In this study the functional role of OPN in human pancreatic cancer and its potential use as a disease marker were analyzed. By real time quantitative PCR, there was a 2.2-fold and 1.6-fold increase of OPN mRNA in pancreatic cancers (n=23) and chronic pancreatitis samples (n=22), respectively, compared to normal pancreatic tissues (n=20). Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumours and in 72% of the lymph node and liver metastases. ELISA analysis of serum samples obtained from pancreatic cancer patients (n=70), chronic pancreatitis patients (n=12), and healthy donors (n=20) showed a 1.6-fold increase in OPN serum levels in patients with tumours and a 1.9–fold increase in patients with chronic pancreatitis. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down-regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. In conclusion, OPN serum levels in pancreatic cancer and chronic pancreatitis patients are not significantly different, thereby restricting its role as a prognostic or follow-up marker. Our results do suggest, however, that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.

ALKYLPHOSPHOCHOLINES - INFLUENCE OF STRUCTURAL VARIATION ON BIODISTRIBUTION AT ANTINEOPLASTICALLY ACTIVE CONCENTRATIONS

SummaryHexadecylphosphocholine (HPC) and octadecylphosphocholine (OPC) show very potent antitumor activity against autochthonous methylnitrosourea-induced mammary carcinomas in rats. The longer-chain and unsaturated homologue erucylphosphocholine (EPC) forms lamellar structures rather than micelles, but nonetheless exhibits antineoplastic activity. Methylnitrosourea was used in the present study to induce autochthonous mammary carcinomas in virgin Sprague-Dawley rats. At 6 and 11 days following oral therapy, the biodistribution of HPC, OPC and EPC was analyzed in the serum, tumor, liver, kidney, lung, small intestine, brain and spleen of rats by high-performance thin-layer chromatography. In contrast to the almost identical tumor response noted, the distribution of the three homologues differed markedly. The serum levels of 50 nmol/ml obtained for OPC and EPC were much lower than the value of 120 nmol/ml measured for HPC. Nevertheless, the quite different serum levels resulted in similar tumor concentrations of about 200 nmol/g for all three of the compounds. Whereas HPC preferably accumulated in the kidney (1 μmol/g), OPC was found at increased concentrations (400 nmol/g) in the spleen, kidney and lung. In spite of the high daily dose of 120 μmol/kg EPC as compared with 51 μmol/kg HPC or OPC, EPC concentrations (100–200 nmol/g) were low in most tissues. High EPC concentrations were found in the small intestine (628 nmol/g). Values of 170 nmol/g were found for HPC and OPC in the brain, whereas the EPC concentration was 120 nmol/g. Obviously, structural modifications in the alkyl chain strongly influence the distribution pattern of alkylphosphocholines in animals. Since EPC yielded the highest tissue-to-serum concentration ratio in tumor tissue (5.1) and the lowest levels in other organs, we conclude that EPC is the most promising candidate for drug development in cancer therapy.

Alkyl phosphocholines: Toxicity and anticancer properties

The study reports on the investigation of acute and subacute toxicity and on antineoplastic activity of hexadecylphosphocholine (HPC), the first compound of a new class of antineoplastic chemotherapeutics. In rats, the LD50 of HPC was 606 μmol/kg; the maximum tolerable dose over four weeks was 39 μmol/kg. Symptoms of toxicity were enteritis, spider cell activation in the liver, hemosiderosis in the spleen and reversible transaminase increase. The best therapeutic effect was observed on methylnitrosourea (MNU)-induced mammary carcinoma in the rat. Two transplantable mammary carcinomas in the rat and autochthonous benzo(a)pyrene-induced sarcomas exhibited low-grade sensitivity to HPC. The MXT mammary carcinoma of the mouse, the Walker 256 carcinosarcoma of the rat, and autochthonous acetoxymethylmethylnitrosamine-induced colonic tumors of the rat were not chemosensitive to HPC.

Imaging anti-angiogenic treatment response with DCE-VCT, DCE-MRI and DWI in an animal model of breast cancer bone metastasis

As current classification systems for the assessment of treatment response in bone metastasis do not meet the needs of oncologists, new imaging biomarkers are desirable. Therefore, the diagnostic impact of dynamic contrast enhanced (DCE)-volumetric computed tomography (VCT) (descriptive analysis), DCE-MRI (two-compartment model) and diffusion weighted imaging (DWI) for monitoring anti-angiogenic therapy effects of the VEGF antibody bevacizumab in breast cancer bone metastases in rats was studied. Nude rats (n=8 animals treated with bevacizumab and n=9 untreated control rats) with site-specific osteolytic bone metastasis of the hind leg were imaged with a 1.5T clinical MRI-scanner in an animal coil as well as in a volumetric CT-scanner at days 30, 40, 50 and 60 after inoculation of MDA-MB-231 human breast cancer cells. From these data, osteolytic lesion size (OLS), peak enhancement (PE), area under the curve (AUC), amplitude (A), exchange rate constant (k(ep)) and apparent diffusion coefficient (ADC) were determined in bone metastases. Prior to changes in OLS (p< or =0.05 at days 50 and 60) there was already a significant decrease in PE, AUC and A (p< or =0.05 at days 40-60) in treated animals compared to controls. However, for k(ep) and ADC there were no significant differences between the groups at any time point (p>0.05 at days 40-60). In conclusion, anti-angiogenic treatment response in osteolytic breast cancer bone metastases can be assessed early with surrogate markers of vascularization, while DWI appears to be insensitive.

Human urinary bladder carcinoma cell lines respond to treatment with alkylphosphocholines

Alkylphosphocholines (APC) constitute a new group of antineoplastic agents without haematological toxicity. Their first clinically available derivative hexadecylphosphocholine (miltefosine) is locally used to control skin metastases of breast cancer. Since intravesical chemotherapy represents a form of topical treatment we investigated whether a new APC with a long alkyl chain would be active against 5637 and EJ bladder cancer cell lines. Their antineoplastic activity was inversely related to the alkyl chain length of the respective APC. Erucylphosphocholine and its congener with modified phosphocholine head erucylphospho-N,N,N-trimethylpropanolamine were the most effective derivatives. APC with alkyl chains over 16 carbons in length induced programmed cell death in both cell lines, as determined by oligonucleosomal DNA fragmentation and morphology. The distinct antineoplastic effects lead us to predict that urinary bladder instillation of APC will be of therapeutic benefit for patients with urinary bladder neoplasia.

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti‐leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL‐60, K‐562, Reh, MOLT‐4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML‐derived K‐562 cell line. Flow cytometry of HL‐60 cells showed a significant decrease of cells in S phase and the formation of a sub‐G1 fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL‐60 cells after 14‐hr exposure. Colony formation by K‐562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long‐term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl‐[2‐(N‐methylpiperidino)‐ethyl]‐phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo,which is unusual for the majority of classical DNA‐interacting anti‐cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti‐leukemic effects, which lack hematological toxicity. Int. J. Cancer,77:778–786, 1998.