Engineering Lipusu with lysophosphatidylcholine for improved tumor cellular uptake and anticancer efficacy.

Liposomes have been developed as drug delivery carriers to enhance the antitumor efficiency of therapeutic agents. Lipusu® (Lip), a paclitaxel (PTX) liposome, has been widely used in the treatment of breast cancer. Compared with PTX, Lip could change the biodistribution and reduce the systemic toxicity. However, there was no positive effect on the entry of PTX into tumor cells, and thus the therapeutic effect was not significantly improved. Therefore, it is meaningful to engineer Lip for improving tumor cellular uptake efficiency. Here, lysophosphatidylcholine (LPC)-engineered Lip (LPC-Lip) was constructed via inserting single chain lipid tails into liposomal lipid bilayers, which was realized by simple incubation. Compared with Lip, the better cellular uptake of liposomes modified with LPC resulted in enhanced cytotoxic activity of LPC-Lip in 4T1 cells. Furthermore, stronger tumor growth inhibition was observed in LPC-Lip treated 4T1 tumor-bearing mice without significant side effects. In conclusion, by modulating the lipid composition of Lip, the antitumor efficacy can be improved, and LPC engineered Lip may serve as a promising formulation of PTX for future cancer therapy.

Stearoyl lysophosphatidylcholine inhibits LPS-induced extracellular release of HMGB1 through the G2A/calcium/CaMKKß/AMPK pathway.

Stearoyl lysophosphatidylcholine (sLPC) has protective effects against several lethal sepsis models, even after induction of sepsis, which is associated with sLPC-mediated inhibition of high mobility group box 1 (HMGB1) release. This study investigated the mechanism by which sLPC inhibits lipopolysaccharide (LPS)-induced extracellular secretion of HMGB1 after the onset of sepsis. sLPC increased AMPK phosphorylation and the binding of AMPK to calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß), one of the upstream signals of AMPK. Inhibition of CaMKKß activity decreased sLPC-mediated inhibition of HMGB1 release, and sLPC increased the concentration of intracellular calcium. Blocking of the macrophage G protein-coupled receptor G2A (G2A) suppressed AMPK phosphorylation, suppressed increases in the intracellular levels of calcium, and prevented the inhibition of HMGB1 release by sLPC. In particular, when macrophages were incubated with sLPC even after LPS treatment, sLPC increased the phosphorylation of AMPK and the binding of CaMKKß and AMPK, and suppressed the secretion of HMGB1. In addition, sLPC administered 1 h before or 4 h after establishment of sepsis significantly diminished circulating HMGB1 levels in mice. sLPC inhibited LPS-induced extracellular release of HMGB1 through the activation of the G2A/calcium/CaMKKß/AMPK pathway. These findings suggest that sLPC may be a potential anti-inflammatory agent for acute inflammatory conditions such as sepsis.

Efficacy of Lysophosphatidylcholine in Combination with Antimicrobial Agents against Acinetobacter baumannii in Experimental Murine Peritoneal Sepsis and Pneumonia Models.

Immune response stimulation to prevent infection progression may be an adjuvant to antimicrobial treatment. Lysophosphatidylcholine (LPC) is an immunomodulator involved in immune cell recruitment and activation. In this study, we aimed to evaluate the efficacy of LPC in combination with colistin, tigecycline, or imipenem in experimental murine models of peritoneal sepsis and pneumonia. We used Acinetobacter baumannii strain Ab9, which is susceptible to colistin, tigecycline, and imipenem, and multidrug-resistant strain Ab186, which is susceptible to colistin and resistant to tigecycline and imipenem. Pharmacokinetic and pharmacodynamic parameters for colistin, tigecycline, and imipenem and the 100% minimal lethal dose (MLD100) were determined for both strains. The therapeutic efficacies of LPC, colistin (60 mg/kg of body weight/day), tigecycline (10 mg/kg/day), and imipenem (180 mg/kg/day), alone or in combination, were assessed against Ab9 and Ab186 at the MLD100 in murine peritoneal sepsis and pneumonia models. The levels of pro- and anti-inflammatory cytokines, i.e., tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), were determined by enzyme-linked immunosorbent assay (ELISA) for the same experimental models after inoculating mice with the MLD of both strains. LPC in combination with colistin, tigecycline, or imipenem markedly enhanced the bacterial clearance of Ab9 and Ab186 from the spleen and lungs and reduced bacteremia and mouse mortality rates (P < 0.05) compared with those for colistin, tigecycline, and imipenem monotherapies. Moreover, at 4 h post-bacterial infection, Ab9 induced higher TNF-α and lower IL-10 levels than those with Ab186 (4 µg/ml versus 3 µg/ml [P < 0.05] and 2 µg/ml versus 3.4 µg/ml [P < 0.05], respectively). LPC treatment combined with colistin, tigecycline, or imipenem modestly reduced the severity of infection by A. baumannii strains with different resistance phenotypes compared to LPC monotherapy in both experimental models.

Choline Supplementation With a Structured Lipid in Children With Cystic Fibrosis: A Randomized Placebo-Controlled Trial.

BACKGROUND: Choline depletion is seen in cystic fibrosis (CF) and pancreatic insufficiency in spite of enzyme treatment and may result in liver, fatty acid, and muscle abnormalities. This study evaluated the efficacy and safety of an easily absorbed choline-rich structured lipid (LYM-X-SORB™ [LXS]) to improve choline status. METHODS: Children with CF and pancreatic insufficiency were randomized to LXS or placebo in a 12-month double blind trial. Dietary choline intake, plasma cholines, plasma and fecal phospholipids, coefficient of fat absorption, pulmonary function, growth status, body composition, and safety measures were assessed. Magnetic resonance spectroscopy for calf muscle choline and liver fat were assessed in a subgroup and compared with a healthy comparison group matched for age, sex, and body size. RESULTS: A total of 110 subjects were enrolled (age 10.4 ±â€Š3.0 years). Baseline dietary choline, 88% recommended, increased 3-fold in the LXS group. Plasma choline, betaine, and dimethylglycine increased in the LXS but not placebo (P = 0.007). Plasma lysophosphatidylcholine and phosphatidylcholine increased, and fecal phosphatidylcholine/phosphatidylethanolamine ratio decreased (P ≤ 0.05) in LXS only, accompanied by a 6% coefficient of fat absorption increase (P = 0.001). Children with CF had higher liver fat than healthy children and depleted calf muscle choline at baseline. Muscle choline concentration increased in LXS and was associated with improvement in plasma choline status. No relevant changes in safety measures were evident. CONCLUSIONS: LXS had improved choline intake, plasma choline status, and muscle choline stores compared with placebo group. The choline-rich supplement was safe, accepted by participants, and improved choline status in children with CF.

Vinyl sulfone analogs of lysophosphatidylcholine irreversibly inhibit autotaxin and prevent angiogenesis in melanoma.

Autotaxin (ATX) is an enzyme discovered in the conditioned medium of cultured melanoma cells and identified as a protein that strongly stimulates motility. This unique ectonucleotide pyrophosphatase and phosphodiesterase facilitates the removal of a choline headgroup from lysophosphatidylcholine (LPC) to yield lysophosphatidic acid (LPA), which is a potent lipid stimulator of tumorigenesis. Thus, ATX has received renewed attention because it has a prominent role in malignant progression with significant translational potential. Specifically, we sought to develop active site-targeted irreversible inhibitors as anti-cancer agents. Herein we describe the synthesis and biological activity of an LPC-mimetic electrophilic affinity label that targets the active site of ATX, which has a critical threonine residue that acts as a nucleophile in the lysophospholipase D reaction to liberate choline. We synthesized a set of quaternary ammonium derivative-containing vinyl sulfone analogs of LPC that function as irreversible inhibitors of ATX and inactivate the enzyme. The analogs were tested in cell viability assays using multiple cancer cell lines. The IC50 values ranged from 6.74 to 0.39 µM, consistent with a Ki of 3.50 µM for inhibition of ATX by the C16H33 vinyl sulfone analog CVS-16 (10b). A phenyl vinyl sulfone control compound, PVS-16, lacking the choline-like quaternary ammonium mimicking head group moiety, had little effect on cell viability and did not inhibit ATX. Most importantly, CVS-16 (10b) significantly inhibited melanoma progression in an in vivo tumor model by preventing angiogenesis. Taken together, this suggests that CVS-16 (10b) is a potent and irreversible ATX inhibitor with significant biological activity both in vitro and in vivo.

Therapeutic efficacy of lysophosphatidylcholine in severe infections caused by Acinetobacter baumannii.

Due to the significant increase in antimicrobial resistance of Acinetobacter baumannii, immune system stimulation to block infection progression may be a therapeutic adjuvant to antimicrobial treatment. Lysophosphatidylcholine (LPC), a major component of phospholipids in eukaryotic cells, is involved in immune cell recruitment and modulation. The aim of this study was to show if LPC could be useful for treating infections caused by A. baumannii. A. baumannii ATCC 17978 was used in this study. Levels of serum LPC and levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1ß, and IL-10 were determined by spectrophotometric assay and enzyme-linked immunosorbent assay (ELISA), respectively, using a murine peritoneal sepsis model in which mice were inoculated with 5.3 log CFU/ml of A. baumannii. The therapeutic efficacy of LPC against A. baumannii in murine peritoneal sepsis and pneumonia models was assessed for 48 h after bacterial infection. At early time points in the murine model of peritoneal sepsis caused by A. baumannii, LPC was depleted and was associated with an increase of inflammatory cytokine release. Preemptive therapy with LPC in murine peritoneal sepsis and pneumonia models markedly enhanced spleen and lung bacterial clearance and reduced the numbers of positive blood cultures and the mouse mortality rates. Moreover, treatment with LPC reduced proinflammatory cytokine production. These data demonstrate that LPC is efficacious as a preemptive treatment in experimental models of peritoneal sepsis and pneumonia caused by A. baumannii.

Docosahexaenoic- and eicosapentaenoic acid-bound lysophospholipids are more effective in suppressing angiogenesis than conjugated docosahexaenoic acid.

Suppression of leukemia, colon cancer, myeloma, and fibrosarcoma to some extent by omega 3 fatty acid bound phospholipids has been reported in the last two decade. However, the anti-angiogenic activity of those phospholipids is still not known. Four kinds of marine phospholipid molecular species i.e. starfish EPA bound diacyl phospholipid (EPA-PC), EPA bound monoacyl phospholipid (EPA-LPC) which was prepare via Lipozyme RMIM mediated partial hydrolysis of EPA-PC, squid DHA bound diacyl phospholipid (DHA-PC), and DHA bound monoacyl phospholipid (DHA-LPC) which was also prepare via Lipozyme RMIM mediated partial hydrolysis of DHA-PC, were subjected to antiangiogenic activity assay by using a piece of rat main artery and a human umbilical cord vein endothelial cell. The lengths of micro vein generated from those tissues after incubation with the above four kinds of phospholipid molecular species were measured and compared. EPA-LPC and DHA-LPC showed strong antiangiogenic activity on the rat main artery tissue, while on the human umbilical cord vein endothelial cells, 100 µM of EPA-LPC in the culture medium, exhibited the most effective suppression on angiogenesis, followed by 100 µM of DHA-LPC. It was concluded that EPA-LPC obtained via Lipozyme RMIM mediated partial hydrolysis of EPA-PC is the most effective omega 3 phospholipid on anti-angiogenesis.

Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of gram-negative and gram-positive shock.

1. Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA). 2. Rats were subjected to (i) endotoxaemia (LPS 6 mg kg(-1) i.v.) and treated with sLPC (1-100 mg kg(-1)), (ii) endotoxaemia and treated with nLPC (10 mg kg(-1)) or (iii) gram-positive shock (PepG 10 mg kg(-1) and LTA 3 mg kg(-1) i.v.) and treated with sLPC (10 mg kg(-1)). 3. Endotoxaemia or gram-positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury. 4. Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1-10 mg kg(-1)) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg(-1)) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by gram-positive shock. nLPC was found to be protective in endotoxaemic animals. 5. The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin-1beta (IL-1beta). 6. In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL-1beta levels caused by gram-negative or gram-positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies.

Suppression of HMGB1 release by stearoyl lysophosphatidylcholine:an additional mechanism for its therapeutic effects in experimental sepsis.

Stearoyl lysophosphatidylcholine (LPC) has recently been proven protective against lethal sepsis by stimulating neutrophils to eliminate invading pathogens through an H2O2-dependent mechanism. Here, we demonstrate that stearoyl LPC, but not caproyl LPC, significantly attenuates circulating high-mobility group box 1 (HMGB1) levels in endotoxemia and sepsis by suppressing endotoxin-induced HMGB1 release from macrophages/monocytes. Neutralizing antibodies against G2A, a potential cell surface receptor for LPC, partially abrogated stearoyl LPC-mediated suppression of HMGB1 release. Thus, stearoyl LPC confers protection against lethal experimental sepsis partly by facilitating the elimination of the invading pathogens and partly by inhibiting endotoxin-induced release of a late proinflammatory cytokine, HMGB1.

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH.

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.

Therapeutic effects of lysophosphatidylcholine in experimental sepsis.

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H(2)O(2) production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-alpha (TNF-alpha) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections.

Studies of the antitumor activity of (2-alkoxyalkyl)- and (2-alkoxyalkenyl)phosphocholines.

Analogues of the synthetic antitumor phospholipid ALP (1-octadecyl-2-methyl-sn-glycero-3-phosphocholine; alkyl lysophospholipid) in which the 1-ether oxygen atom has been removed have been prepared and evaluated for in vitro and in vivo anticancer activity. Compounds are presented in which the saturated long chain varies in length from 8 to 25 carbon atoms. Sites of unsaturation are also incorporated into the framework in some examples. In particular, rac-(2-ethoxyeicosyl)phosphocholine (10) displays the best in vivo activity of the chemical series against a variety of transplanted tumors and activates murine peritoneal macrophages to express tumor cytotoxicity in vitro. However, 10 does not offer the wide spectrum of antitumor activity we feel necessary to warrant further study.

Ether lipid derivatives: antineoplastic activity in vitro and the structure-activity relationship.

The antineoplastic activity of two ether lipid derivatives, the alkyl-lysophospholipid derivative (ALP) ET-18-OCH3 and the ether-linked lipoidal amine CP-46,665 was tested in a human tumor clonogenic assay (HTCA) in vitro. CP-46,665 suppressed the colony formation of various human tumors with a slight dose response relation after 1 hr incubation and with a clear optimum (85% response rate) after continuous exposure in the higher dose range tested (10 micrograms/ml). ET-18-OCH3 did not have substantial activity after 1 hr of incubation. However, when continuous exposure to the compound was used, ET-18-OCH3 seemed to have a modest dose response effect and yielded a response in about 60% of the tumor cell samples tested in the higher dose range (10 micrograms/ml). Thus, both compounds have in vitro antitumor activity in the HTCA within a dose range of 1-10 micrograms/ml, especially during continuous exposure. The tumor specific type activity was found in breast cancer, ovarian cancer, lung cancer and mesothelioma. Both compounds caused decreases in colony formation down to the 0%, 2% and 4% levels. In a comparison of specimens in which both compounds were used, only one of five times showed a discordance in sensitivity or resistance; therefore the compounds appear similar in their in vitro activity. In a second set of experiments we tested the structure-activity relationship among a variety of ALP in the [3H]thymidine incorporation assay after incubation with HL-60 leukemic blasts and other neoplastic cells from human origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonogenicity of normal and malignant hematopoietic progenitor cells after exposure to synthetic alkyl-lymphospholipids.

Alkyl-lysophospholipids (ALP) are synthetic analogues of natural lysophosphatidylcholine and represent a new class of anti-tumor agents. They are cytotoxic in vitro with a high selectivity for neoplastic cells which, in contrast to normal cells, lack an alkyl-cleavage enzyme to degrade the adsorbed ALP molecules. As ALP accumulates, it interferes with normal membrane phospholipid turnover and eventually causes disruption of membrane integrity. To evaluate the potential value of ALP in eliminating leukemic cells from remission marrows prior to autologous transplantation, we tested the effect of various ALPs on the clongenicity of normal human marrow cells and on promyelocytic leukemia HL-60. A remarkable difference in the dose response to ALP of normal marrow cells an HL-60 was observed. After an incubation period of 24 h, the same inhibition of clonogenicity in HL-60 occurred at ALP concentrations 4 times lower than in normal marrow cells. Reducing the exposure time to 6 h enhanced the selectivity further: whereas HL-60 colonies were nearly completely inhibited at 16 micrograms ALP/ml, more than 50% of normal CFU-c and BFU-E were recovered after incubation with 48 micrograms/ml. No further increase in selectivity was achieved by changing the incubation temperature. Both thioether- and alkyl-analogues were active and no difference was observed between methoxy- and acylamino-substituted ALPs. We conclude that this selective cytotoxicity makes ALP compounds worth further study as purging agents in autologous bone marrow transplantation programs.

Influence of the alkyllysophospholipid ET-18-OCH3 on methylnitrosourea-induced rat mammary carcinomas.

This study describes the efficacy of the alkyllysophospholipid 1-octadecyl-2-methoxy-Sn-racglycero-3-phosphocholine (ET-18-OCH3) in inhibiting the growth of methylnitrosourea-induced mammary carcinomas in Sprague-Dawley rats. In experiment A 2 X 10 mg/kg Et-18-OCH3 were administered daily for 10 weeks prior to manifestation of mammary carcinomas which resulted in a significant inhibition of median tumor number and median tumor volume per rat. Treatment of established tumors (experiment B) with 6 and 60 mg/kg ET-18-OCH3 daily for 3 weeks effected a stagnation in tumor growth for the higher dosage only with 90% tumor inhibition in comparison to untreated controls; at the same time, however, clear toxic effects were seen, thus indicating a narrow therapeutic index of ET-18-OCH3 in single-drug therapy. Combination of ET-18-OCH3 with compounds possessing a different toxicity spectrum is suggested.

Experimental chemotherapy of radiation injury with synthetic lysophospholipid analogs in mice.

Synthetic lysophospholipids represent a variety of analogs of the naturally occurring 2-lysophosphatidylcholine. Some of these compounds showed significant therapeutic effects on the survival of mice following radiation injury when administered after various doses of whole-body X irradiation. Such therapeutic effects were discernible even when the treatment was given 6 hr after irradiation, and both intravenous and oral application were effective. Intravenous application of 2 X 25 mg/kg lysophospholipid after whole-body X irradiation around the LD50 resulted in significantly higher numbers of surviving animals. The mode of action remains speculative.

Early tumor and leukemia response to alkyllysophospholipids in a phase I study.

In a phase I study on the toxicity and toleration of alkyllysophospholipids, tumor and leukemia responses have been noted in the first treated patients. Six patients with solid malignomas of different histologic types and one patient with acute myeloid leukemia are evaluable so far. All of them suffered from metastatic or wide-spread disease, were refractory to adequate polychemotherapy or other treatment modalities, or have been found untreatable because of poor general condition. Four cases revealed objective tumor and leukemia response with a minor response in a hypernephroma, two partial remissions in nonsmall cell bronchogenic carcinomas and reduction of leukemic blasts to less than 10% in acute myeloid leukemia. Limiting toxicity started with doses of 20 mg/kg given daily showing transient injury of renal and liver functions.

Studies on various parameters influencing leukemic cell destruction by alkyl-lysophospholipids.

Critical parameters of alkyl-lysophospholipid (ALP) induced destruction of freshly isolated human leukemic cells have been evaluated. The destructive activity of ALP is shown to be competitively inhibited by metabolizable lysophospholipids added to the cultures. It has also been found that destruction depends on the amount of serum present. Temperature and Ph strongly influence the cytotoxic activity of ALP. A slight decrease in temperature causes a reduction in cell death, whereas a temperature increase results in a marked potentiation. At low pH ALP cytotoxicity is inhibited. Incubation of cells with combinations of ALP and other cytotoxic drugs revealed a striking cytotoxic synergism with vinca-alkaloids, whereas corticosteroids retarded ALP induced cell destruction.