The pyrimidine analogue gemcitabine (dFdC) is frequently used in the treatment of patients with solid tumors. However, after i.v. application dFdC is rapidly inactivated by metabolization. Here, the potential of thermosensitive liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2-TSL) were investigated as carrier and targeting system for delivery of dFdC in combination with local hyperthermia (HT).

Background Erufosine is a promising anticancer drug that increases the efficacy of radiotherapy in glioblastoma cell lines in vitro . Moreover, treatment of nude mice with repeated intraperitoneal or subcutaneous injections of Erufosine is well tolerated and yields drug concentrations in the brain tissue that are higher than the concentrations required for cytotoxic drug effects on glioblastoma cell lines in vitro . Methods In the present study we aimed to evaluate the effects of a combined treatment with radiotherapy and Erufosine on growth and local control of T98G subcutaneous glioblastoma xenograft- tumours in NMRI nu/nu mice. Results We show that repeated intraperitoneal injections of Erufosine resulted in a significant drug accumulation in T98G xenograft tumours on NMRI nu/nu mice. Moreover, short-term treatment with 5 intraperitoneal Erufosine injections caused a transient decrease in the growth of T98G tumours without radiotherapy. Furthermore, an increased radiation-induced growth delay of T98G xenograft tumours was observed when fractionated irradiation was combined with short-term Erufosine-treatment. However, no beneficial drug effects on fractionated radiotherapy in terms of local tumour control were observed. Conclusions We conclude that short-term treatment with Erufosine is not sufficient to significantly improve local control in combination with radiotherapy in T98G glioblastoma xenograft tumours. Further studies are needed to evaluate efficacy of extended drug treatment schedules.

Background and Purpose The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. Materials and methods Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. Results The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. Conclusions Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.

Background: Alkylphosphocholines represent promising antineoplastic drugs that induce cell death in tumor cells by primary interaction with the cell membrane. Recently we could show that a combination of radiotherapy with Erufosine, a paradigmatic intravenously applicable alkylphosphocholine, in vitro leads to a clear increase of irradiation-induced cell death. In view of a possible combination of Erufosine and radiotherapy in vivo we determined the pharmacokinetics and bioavailability as well as the tolerability of Erufosine in nude mice.Methods: NMRI (nu/nu) nude mice were treated by intraperitoneal or subcutaneous injections of 5 to 40 mg/kg body weight Erufosine every 48 h for one to three weeks. Erufosine-concentrations were measured in brain, lungs, liver, small intestine, colon, spleen, kidney, stomach, adipoid tissue, and muscle by tandem-mass spectroscopy. Weight course, blood cell count and clinical chemistry were analyzed to evaluate general toxicity.Results: Intraperitoneal injections were generally well tolerated in all dose groups but led to a transient loss of the bodyweight (< 10%) in a dose dependent manner. Subcutaneous injections of high-dose Erufosine caused local reactions at the injection site. Therefore, this regimen at 40 mg/kg body weight Erufosine was stopped after 14 days. No gross changes were observed in organ weight, clinical chemistry and white blood cell count in treated compared to untreated controls except for a moderate increase in lactate dehydrogenase and aspartate-aminotransferase after intensive treatment. Repeated Erufosine injections resulted in drug-accumulation in different organs with maximum concentrations of about 1000 nmol/g in spleen, kidney and lungs.Conclusion: Erufosine was well tolerated and organ-concentrations surpassed the cytotoxic drug concentrations in vitro. Our investigations establish the basis for a future efficacy testing of Erufosine in xenograft tumor models in nude mice alone and in combination with chemo-or radiotherapy.

The molecular conformation and packing of three lysophosphatidyl-cholines: 3-palmitoyl-D-glycero-1-phosphocholine (PPC), 3-hexadecyl-D-glycero-1-phosphocholine (HPC) and 3-palmitoyl-DL-glycero-1-phospho-N,N-dimethylethanolamine (PPEM2) have been determinated by X-ray single crystal analyses. PPC crystallizes as the monohydrate and HPC as chloroform solvate, both with triclinic unit cells (space group P1) containing two independent molecules in almost identical packing arrangements. The two molecules of PPC/HPC are mirror image conformers with respect to their head groups and pack separately in either half of a bilayer arrangement with interdigitating hydrocarbon chains and interdigitating head groups. The racemic PPEM2 also crystallizes with a very similar interdigitating packing arrangement. The unit cell, however, is monoclinic (space group P21/a) and comprises four molecules, arranged as pairs of centrosymmetric D/L conformers at either side of the bilayer. In PPC and HPC the C16-hydrocarbon chains interdigitate with an overlap of 13 carbon atoms only, leaving a cavity to accommodate the solvate molecules. The chain matrices have identical tilt (45°), but different chain packing modes (O′⊥ and O∥). In PPEM2 the chain ends penetrate with 18-atoms interdigitation to the glycerol region of oppositely oriented molecules and pack in a hybrid matrix with 37° tilt. Despite the differences in hydrocarbon chain attachment (ester/ether) and degree of N-methylation the structures show great similarities, in particular with respect to the head group conformation, which apparently is favored by intrinsic energetics.

Abstract Background Alkylphosphocholines constitute a novel class of antineoplastic synthetic phospholipid derivatives that induce apoptosis of human tumor cell lines by targeting cellular membranes. We could recently show that the first intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC) is a potent inducer of apoptosis in highly resistant human astrocytoma/glioblastoma cell lines in vitro. ErPC was shown to cross the blood brain barrier upon repeated intravenous injections in rats and thus constitutes a promising candidate for glioblastoma therapy. Aim of the present study was to analyze putative beneficial effects of ErPC and its clinically more advanced derivative erucylphosphohomocholine (erucyl-N, N, N-trimethylpropanolaminphosphate, ErPC3, Erufosine™ on radiation-induced apoptosis and eradication of clonogenic tumor cells in human astrocytoma/glioblastoma cell lines in vitro. Results While all cell lines showed high intrinsic resistance against radiation-induced apoptosis as determined by fluorescence microscopy, treatment with ErPC and ErPC3 strongly increased sensitivity of the cells to radiation-induced cell death (apoptosis and necrosis). T98G cells were most responsive to the combined treatment revealing highly synergistic effects while A172 showed mostly additive to synergistic effects, and U87MG cells sub-additive, additive or synergistic effects, depending on the respective radiation-dose, drug-concentration and treatment time. Combined treatment enhanced therapy-induced damage of the mitochondria and caspase-activation. Importantly, combined treatment also increased radiation-induced eradication of clonogenic T98G cells as determined by standard colony formation assays. Conclusion Our observations make the combined treatment with ionizing radiation and the membrane targeted apoptosis modulators ErPC and ErPC3 a promising approach for the treatment of patients suffering from malignant glioma. The use of this innovative treatment concept in an in vivo xenograft setting is under current investigation.

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8–33% (Apaf-1) and to 30–50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17–33% (Apaf-1) and to 38–50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.

At concentrations effecting apoptosis, the alkylphosphocholine ErPC3 induced increased expression of the Rb protein in breast cancer (MCF-7) and leukemia (SKW-3, AR-230) cell lines as well as hypophosphorylation (K-562, CMLT-1, DOHH-2) and fragmentation of Rb (BV-173, SKW-3) in leukemia cell lines. ErPC3 exerts at least part of its antineoplastic activity by apoptosis, and this chain of events comprises early changes in the lipid raft fraction of the cellular membrane as well as modulation of different signal molecules, such as Abl, Bcr-Abl (fusion protein), and Rb.

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNF α -related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c , activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.

Acanthamoeba keratitis is a corneal disease associated predominantly with contact lens wear. The occurrence of Acanthamoeba keratitis has been rising since 1990 in correlation to the growing number of contact lens wearers. To date approximately 2000 cases have been published around the world. Due to the complicated diagnostics, the elaborate treatment and the usually bad compliance of the patients, Acanthamoeba keratitis unfortunately very often takes a serious progression, which may lead to serious visual loss and perforating keratoplasty. Today, local treatment with a combination of polyhexamethylene biguanide (PHMB) and propamidine isethionate (Brolene) is considered the first line therapy for Acanthamoeba keratitis. Alternatively also a combination of propamidine and chlorhexidine or neomycine achieves good therapeutic results. However, the complicated mode of application consistently remains a problem. The intensive local treatment, i.e. hourly application of therapeutics during the first three days day and night makes hospitalization inevitable. Moreover, sufficient efficacy can not always be achieved, and also resistance against propamidine has already been observed. Recently propamidine has sometimes been replaced by hexamidine, which seems to have a greater cysticidal activity. A new path might be struck by the application of alkylphosphocholines. These are phosphocholines esterified to aliphatic alcohols. They exhibit in vitro and in vivo antineoplastic activity and have been shown to be cytotoxic against Leishmania donovani, Trypanosoma cruzi, and Entamoeba histolytica. A recent study has demonstrated that particularly hexadecylphosphocholine is highly effective also against various strains of Acanthamoeba.

Erucylphosphocholine (ErPC) is a promising candidate for the treatment of human brain tumors. The aim of the present study was to investigate whether structural modifications of ErPC would improve its antineoplastic activity in vitro. The novel alkylphosphocholine (APC) derivatives docosenyl-(cis-10,11)-phosphocholine, tricosenyl-(cis-12,13)-phosphocholine, heneicosenyl-(cis-12,13)-phosphocholine and erucyl-N,N,N-trimethylpropanolaminophosphate all reduced cell growth and viability of rat and human astrocytoma/glioblastoma (AC/GBM) cell lines (C6, T98G, U87MG, A172) and had improved antineoplastic activity when compared to the prototypical APC hexadecylphosphocholine (HePC). However, the four cell lines differed in their sensitivity to the APC derivatives. A172 cells were most sensitive to their cytostatic action and T98G cells to their cytotoxic action. The LC50 values for T98G cells after a 72-h exposure to the novel derivatives varied between 25 and 54 µM compared to 45±8.1 µM for ErPC. Complete killing of T98G cells was obtained with all derivatives at 90 µM. Structural modifications of the chain length of the alcohol moiety as well as changing the position of the double bond within the alkyl chain improved cytotoxicity of the APC in C6 and A172 cells and to a lesser extent in T98G cells, whereas U87MG cells showed almost similar sensitivities to the novel drugs and ErPC. Increasing the distance between the phosphorus and nitrogen atoms within the polar phosphocholine group did not alter antineoplastic activity but modified physicochemical characteristics, e.g. increased the solubility in water. In a similar manner to ErPC, all derivatives induced growth arrest in the G2/M phase of the cell cycle and apoptotic cell death. Importantly, none of the derivatives showed hemolytic activity. As there was no clear superiority of any of the novel derivatives, ErPC remains the leading APC derivative for future clinical trials in brain tumor chemotherapy.

BACKGROUND:Erucylphosphocholine (ErPC) has been shown to exert strong antineoplastic effects against various brain tumor cell lines in vitro. Since ErPC only enters the brain after long-term treatment, ineffective drug delivery to the tumor is considered to be the reason for the moderate responses to chemotherapy with ErPC observed in animal brain tumor models. We investigated a recently described method for chemically opening the blood-brain barrier (BBB) using intraarterial administration of alkylglycerols to increase the transfer of ErPC into the brain.METHODS:ErPC (40 mg/kg) was given to C6 glioma-bearing rats either as a single intracarotid bolus injection in the presence or absence of 1- O-pentylglycerol (300 m M) or as an intracarotid infusion in conjunction with bradykinin. Brain tissue concentrations were analyzed and compared to values obtained after intravenous ErPC treatment over 14 and 30 days (cumulative ErPC doses of 210 and 350 mg/kg, respectively).RESULTS:Pentylglycerol-induced BBB opening resulted in a significant increase in ErPC delivery to the tumor (17-fold) and, to a lesser extent, to the surrounding ipsilateral brain (7-fold) compared to intraarterial ErPC administration without alkylglycerol ( P<0.05). Furthermore, the resulting ErPC concentrations in the brain tumor exceeded those obtained in tumor and tumor-free brain after long-term intravenous ErPC administration. In contrast to this, intracarotid bradykinin did not increase the transfer of ErPC to the tumor or tumor-free brain.CONCLUSIONS:The intracarotid administration of pentylglycerol represents a novel and nontoxic method of overcoming the limited access of ErPC to both brain tumors and brain tissue adjacent to tumors. The present results provide further evidence that chemical opening of the BBB by intraarterial alkylglycerols is a promising new concept for improving delivery of chemotherapeutic agents to brain tumors.

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.

The aim of this study was to enhance the antileukemic efficacy of the alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) in chronic myeloid leukemia (CML)-derived cell lines by a bcr-directed antisense oligonucleotide (ASO-bcr). The mechanism was substantiated by Western blotting of the BCR-ABL expression level of CML cells, and the efficacy was substantiated by inhibition of colony formation compared with normal hematopoietic cells. The clonogenicity of K-562 cells expressing high levels of p210(BCR-ABL) was inhibited significantly by the ASO-bcr (T/C%, 30; P < 0.05) but not by ErPC3 (T/C%, 70). Combined sequential exposure to ErPC3 and the ASO-bcr, however, inhibited synergistically colony growth (T/C%, 3; P < 0.01). The colony growth of BV-173 cells expressing lower levels of p210(BCR-ABL) than K562 cells was inhibited to a greater extent by the ASO-bcr (T/C%, 15; P < 0.01). AR-230 cells that express high levels of p230(BCR-ABL) showed an intermediate decrease in colony formation in response to the ASO-bcr (T/C%, 20; P < 0.05). BCR-ABL levels of BV-173, CML-T1, and LAMA-84 cells were reduced in response to the ASO-bcr, as evidenced by Western blot. However, K-562 and AR-230 cells showed reduced BCR-ABL expression only after repeated treatment. ErPC3 and the ASO-bcr did not reduce colony formation (CFU-GM) of normal mouse bone marrow cells from long-term bone marrow cell cultures; instead, ErPC3 stimulated colony formation (P < 0.05) and did not induce chromosomal aberrations in mouse bone marrow. In conclusion, the combination of ErPC3 with a suitable antisense oligonucleotide inhibited synergistically colony formation of CML cell lines without damaging normal cells and thus might have a bearing on the purging of autologous hematopoietic transplants in CML patients.

The blood-brain barrier hinders progress in the chemotherapy of brain tumors due to insufficient penetration of anticancer drugs into the brain tissue. Short-chain alkylglycerols affect the physicochemical properties of biological membranes. The enhancement of the blood-brain barrier permeability by intra-arterial administration of alkylglycerols was investigated in tumor-free and C6 astroglioma bearing rats. The antineoplastic agents cisplatin and methotrexate and the antibiotics vancomycin and gentamicin were selectively injected into the right internal carotid artery in the absence and presence of various alkylmono-, alkyldi-, and alkyltriglycerols. In normal rats the intra-arterial administration of the drugs without alkylglycerols resulted in low drug concentrations in brain tissue. In the presence of alkylglycerols (0.01-0.3 M) a reversible (within minutes) and concentration-dependent enrichment of the coinjected agents was found, preferentially in the ipsilateral hemisphere. The extent of drug accumulation in the brain was modified by changes in the chemical structure of the alkylglycerols. The effect increased with the chain length of the alkyl group, decreased with the number of glycerols, and varied from 2- to more than 230-fold compared to controls. In rats with C6 tumors 1-O-pentylglycerol increased the delivery of methotrexate 18-fold in the tumor, 28-fold in the surrounding brain, 18-fold in the contralateral brain, and 19-fold in the cerebellum compared to controls with methotrexate in the absence of pentylglycerol. In conclusion, the intra-arterial administration of alkylglycerols represents a novel and well controllable method for enhanced drug delivery to the brain and to brain tumors.

The clinical use of alkylphosphocholines (APC) in cancer patients is restricted because of the high gastrointestinal toxicity and the need for oral administration. Therefore we evaluated the clinical pharmacology of erucylphosphocholine (ErPC), the first derivative of the APC family suitable for intravenous administration with strong antineoplastic activity, in vitro and in vivo in rats. The pharmacokinetic parameters after a single intravenous dose of 40 mg/kg were calculated using a two-compartment model: Cmax= 1.6 ± 0.3 μmol/ml, T1/2α= 0.18 ± 0.09 h, T1/2β= 3.3 ± 0.88 h, clearance = 9.7 ± 1.2 ml/h, AUC = 2.5 ± 0.3 μmol/ml per h and Vss = 40.4 ± 7.9 ml. Biodistribution studies were performed after repeated ErPC administration at different doses. Intravenous injections of 20 mg/kg given at intervals of 48 h for up to 4 weeks were well tolerated. Neither clinical evaluation nor laboratory parameters (haematology and clinical chemistry) revealed toxic side effects. In contrast, higher doses of ErPC (40 mg/kg per 48 h) led to weight loss. After 2 and 4 weeks of therapy with 20 mg/kg per 48 h a high ErPC accumulation was found in the adrenal glands, small intestine and brain. The brain to serum concentration ratios averaged 2.1 after 2 weeks and 4.5 after 4 weeks. Significant leucocytosis and thrombocytosis were observed after 4 weeks of ErPC treatment. The findings suggest that ErPC is a suitable candidate for clinical trials. In particular, owing to the high accumulation in brain tissue, ErPC is a potential agent for chemotherapy against malignant brain tumours.

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.

We show for the first time that secreted low-molecular weight phospholipase A2 (EC 3.1.1.4) catalyzes the deacylation of monoacylphosphoglycerides directly from the sn-1 position, although at a very low rate: purified phospholipase A2 enzymes from bee venom, crotalus atrox venom, and porcine pancreas hydrolyze the sn-1 ester bond in 1-palmitoyl-2-O-methyl-sn-glycero-3-phosphorylcholine. Hydrolytic rates with the corresponding isomer, 1-O-methyl-2-palmitoyl-sn-glycero-3-phosphorylcholine, are about 3-4 orders of magnitude higher. The similarities in Ca2+ requirement and inactivation profiles suggest that deacylation, albeit with different rates, from both sn-1 and sn-2 positions is catalyzed by the same catalytic site of phospholipase A2. Furthermore, evidence is provided that phospholipase A2-catalyzed 1-acyl lysophospholipid deacylation is mediated by sn-1-directed action, but above pH 7 acyl migration with subsequent enzyme-catalyzed hydrolytic cleavage from the sn-2 position contributes to the overall deacylation of monoacylphosphoglycerides, acyl migration becoming eventually the rate-limiting factor.

The conformation of hexadecylphosphocholine (HPC) and its interaction with model membranes prepared from dipalmitoylphosphatidylcholine (DPPC) have been investigated by molecular dynamics (MD) and NMR methods. The results of MD studies indicate that in the global minimum energy structure, the phosphocholine head group of the drug is bent at τ18 and τ19, while the hexadecyl chain is nearly extended. In the two-dimensional nuclear Overhauser effect (NOE) experiments, intramolecular NOEs have been observed between the N-methyl moieties of the head group and the methylene protons of the alkyl chain. Such a folding of the molecule is seen in several of the local minimum energy structures computed by MD simulations. The interaction of HPC with DPPC as studied by 31P NMR shows a strong interaction between head groups of the two molecules.