Optimization of Alkyldiethanolamides Synthesis from Terminalia catappa L. Kernel Oil through Enzymatic Reaction.

Alkyldiethanolamides (fatty acid diethanolamides) synthesis from Terminalia catappa L. kernel oil was optimized using lypozyme as a catalyst. The result showed that the optimal reaction conditions were 2 hours reaction time, with a ratio of oil mass (g) to diethanolamine (mmol) of 1:5, a ratio of oil mass to enzyme (g) of 1: 0.075, and a temperature of 40°C. The percentage of alkyldiethanolamides at optimum condition was 56-60%. The synthesis results were also analyzed by FTIR. FTIR spectra revealed specific absorption at several wave numbers (3434 cm-1, 1655 cm-1, 1280 cm-1), indicating that amide and alcohol bonds (C=O, C-N, and O-H) were formed. GC-MS was employed to identify the types of fatty acid diethanolamides that were successfully synthesized. The fatty acid diethanolamides formed were palmitoyldiethanolamide (Rt = 32.96 min) and oleyldiethanolamide (Rt = 35.57 min). The total nitrogen content of alkyldietanolamides was 0.26%, or 0.19 mmol of the amide group in 1 g of sample.

Lactose oleate as new biocompatible surfactant for pharmaceutical applications.

Sugar fatty acid esters are an interesting class of non-ionic, biocompatible and biodegradable sugar-based surfactants, recently emerged as a valid alternative to the traditional commonly employed (e.g. polysorbates and polyethylene glycol derivatives). By varying the polar head (carbohydrate moiety) and the hydrophobic tail (fatty acid), surfactants with different physico-chemical characteristics can be easily prepared. While many research papers have focused on sucrose derivatives, relatively few studies have been carried out on lactose-based surfactants. In this work, we present the synthesis and the physico-chemical characterization of lactose oleate. The new derivative was obtained by enzymatic mono-esterification of lactose with oleic acid. Thermal, surface, and aggregation properties of the surfactant were studied in detail and the cytotoxicity profile was investigated by MTS and LDH assays on intestinal Caco-2 monolayers. Transepithelial electrical resistance (TEER) measurements on Caco-2 cells showed a transient and reversible effect on the tight junctions opening, which correlates with the increased permeability of 4 kDa fluorescein-labelled dextran (as model for macromolecular drugs) in a concentration dependent manner. Moreover, lactose oleate displayed a satisfactory antimicrobial activity over a range of Gram-positive and Gram-negative bacteria. Overall, the obtained results are promising for a further development of lactose oleate as an intestinal absorption enhancer and/or an alternative biodegradable preservative for pharmaceutical and food applications.

Synthesis and biological evaluation of novel lipoamino acid derivatives.

Seven novel lipoamino acid conjugates were synthesized from methyl oleate and amino acids. Methyl oleate was grafted to different amino acids using thioglycolic acid as a spacer group. Seven derivatives (3a-g) were prepared and characterized by spectral data (NMR, IR and MS spectral studies). All the derivatives were studied for their antimicrobial, anti-biofilm and anticancer activities. Among all the derivatives, it was found that compound 3b was the most potent antibacterial compound which showed good activity against four Gram positive bacterial strains and also exhibited excellent antifungal activity against a fungal strain. In the anti-biofilm assay, compound 3b showed promising activity with IC50 value of 2.8µM against Bacillus subtilis MTCC 121. All the compounds showed anticancer activities with 3c showing promising anticancer activity (IC50=15.3-22.4µM) against the four cell lines tested.

Quantitative Structure-Cytotoxicity Relationship of Oleoylamides.

Eighteen oleoylamides were subjected to quantitative structure-activity relationship analysis based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to assess their biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell, oral keratinocyte, primary gingival epithelial cells) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-selectivity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal human oral cells to that against OSCC cell lines. Potency-selectivity expression (PSE) was determined by the ratio of TS to CC50 against OSCC. Anti-HIV activity was evaluated by the ratio of CC50 to the concentration leading to 50% cytoprotection from HIV infection (EC50). Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method. Among 18 derivatives, compounds 8: with a catechol group) and 18: with a (2-pyridyl)amino group) had the highest TS. On the other hand, doxorubicin and 5-fluorouracil (5-FU) were more highly cytotoxic to normal epithelial cells, displaying unexpectedly lower TS and PSE values. None of the compounds had anti-HIV activity. Among 330 chemical descriptors, 75, 73 and 19 descriptors significantly correlated to the cytotoxicity to normal and tumor cells, and TS, respectively. Multivariate statistics with chemical descriptors for molecular polarization and hydrophobicity may be useful for the evaluation of cytotoxicity and TS of oleoylamides.

Synthesis of oleoylethanolamide using lipase.

An effective process for the enzymatic synthesis of oleoylethanolamide is described in this study. The process included purification of a commercial oleic acid product and then optimization of the reaction between the purified oleic acid and ethanolamine in the presence of hexane and a lipase. Under the optimal amidation reaction conditions identified, oleoylethanolamide was obtained with 96.6% purity. The synthesis was also conducted on a large scale (50 mmol of each of the reactants), and oleoylethanolamide purity and yield after crystallization purification were 96.1 and 73.5%, respectively. Compared to the previous studies, the current method of preparing high-purity oleoylethanolamide is more effective and economically feasible. The scalability and ease for such synthesis make it possible to study the biological and nutritional functions of the cannabinoid-like oleoylethanolamide in animal or human subjects.

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery.

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.

Preparation and comparison of cytotoxic complexes formed between oleic acid and either bovine or human α-lactalbumin.

α-Lactalbumin is a ubiquitous calcium-binding milk protein with a well-characterized function in regulating the synthesis of lactose. An entirely different activity has been shown to occur when a complex is formed between calcium-free α-lactalbumin and oleic acid. This complex shows strong cytotoxic action against several cancer cells, and several mechanisms have been suggested to account for this cell-killing activity. Most studies have been performed using the human protein, but bovine α-lactalbumin shows similar activity. A new and simple 2-step method for purification of calcium-free α-lactalbumin has been developed, and the resulting highly purified preparation was used to generate a complex with oleic acid. Using 3 different cell lines and 2 types of cell viability assays, the bovine and human α-lactalbumin showed comparable cytotoxic activity. The effect was apparent after 15 min of incubation and was inhibited by the presence of fetal bovine serum or bovine serum albumin. The bovine protein might be a useful alternative to the human protein, but also raises the question whether cytotoxic activity could be generated in different kinds of food containing α-lactalbumin.

Inhibition of human leukocyte elastase, plasmin and matrix metalloproteinases by oleic acid and oleoyl-galardin derivative(s).

Molecular modeling was undertaken at aims to analyze the interactions between oleic acid and human leukocyte elastase (HLE), plasmin and matrix metalloproteinase-2 (MMP-2), involved in the inhibitory capacity of fatty acid towards those proteases. The carboxylic acid group of the fatty acid was found to form a salt bridge with Arg(217) of HLE while unsaturation interacted with Phe(192) and Val(216) at the S(3) subsite, and alkyl end group occupied S(1) subsite. In keeping with the main contribution of kringle 5 domain in plasmin-oleic acid interaction [Huet E et al. Biochem Pharmacol 2004;67(4):643-54], docking computations revealed that the long alkyl chain of fatty acid inserted within an hydrophobic groove of this domain with the carboxylate forming a salt bridge with Arg(512). Finally, blind docking revealed that oleic acid could occupy both S'(1) subsite and Fn(II)(3) domain of MMP-2. Several residues involved in Fn(II)(3)/oleic acid interaction were similarly implicated in binding of this domain to collagen. Oleic acid was covalently linked to galardin (at P'(2) position): OL-GAL (CONHOH) or to its carboxylic acid counterpart: OL-GAL (COOH), with the idea to obtain potent MMP inhibitors able to also interfere with elastase and plasmin activity. OL-GALs were found less potent MMP inhibitors as compared to galardin and no selectivity for MMP-2 or MMP-9 could be demonstrated. Docking computations indicated that contrary to oleic acid, OL-GAL binds only to MMP-2 active site and surprisingly, hydroxamic acid was unable to chelate Zn, but instead forms a salt bridge with the N-terminal Tyr(110). Interestingly, oleic acid and particularly OL-GALs proved to potently inhibit MMP-13. OL-GAL was found as potent as galardin (K(i) equal to 1.8nM for OL-GAL and 1.45nM for GAL) and selectivity for that MMP was attained (2-3 log orders of difference in inhibitory potency as compared to other MMPs). Molecular modeling studies indicated that oleic acid could be accommodated within S'(1) pocket of MMP-13 with carboxylic acid chelating Zn ion. OL-GAL also occupied such pocket but hydroxamic acid did not interact with Zn but instead was located at 2.8Å from Tyr(176). Since these derivatives retained, as their oleic acid original counterpart, the capacity to inhibit the amidolytic activity of HLE and plasmin as well as to decrease HLE- and plasmin-mediated pro MMP-3 activation, they might be of therapeutic value to control proteolytic cascades in chronic inflammatory disorders.

Synthesis and preliminary evaluation of 18-(18)F-fluoro-4-thia-oleate as a PET probe of fatty acid oxidation.

UNLABELLED: Fatty acid oxidation (FAO) is a major energy-providing process with important implications in cardiovascular, oncologic, neurologic, and metabolic diseases. A novel 4-thia oleate analog, 18-(18)F-fluoro-4-thia-oleate ((18)F-FTO), was evaluated in relationship to the previously developed palmitate analog 16-(18)F-fluoro-4-thia-palmitate ((18)F-FTP) as an FAO probe. METHODS: (18)F-FTO was synthesized from a corresponding bromoester. Biodistribution and metabolite analysis studies were performed in rats. Preliminary small-animal PET studies were performed with (18)F-FTO and (18)F-FTP in rats. RESULTS: A practical synthesis of (18)F-FTO was developed, providing a radiotracer of high radiochemical purity (>99%). In fasted rats, myocardial uptake of (18)F-FTO (0.70 +/- 0.30% dose kg [body mass]/g [tissue mass]) was similar to that of (18)F-FTP at 30 min after injection. At 2 h, myocardial uptake of (18)F-FTO was maintained, whereas (18)F-FTP uptake in the heart was 82% reduced. Similar to (18)F-FTP, (18)F-FTO uptake by the heart was approximately 80% reduced at 30 min by pretreatment of rats with the CPT-I inhibitor etomoxir. Folch-type extraction analyses showed 70-90% protein-bound fractions in the heart, liver, and skeletal muscle, consistent with efficient trafficking of (18)F-FTO to the mitochondrion with subsequent metabolism to protein-bound species. Preliminary small-animal PET studies showed rapid blood clearance and avid extraction of (18)F-FTO and of (18)F-FTP into the heart and liver. Images of (18)F-FTO accumulation in the rat myocardium were clearly superior to those of (18)F-FTP. CONCLUSION: (18)F-FTO is shown to be a promising metabolically trapped FAO probe that warrants further evaluation.

Chemical synthesis of 9(Z)-octadecenamide and its hypolipidemic effect: a bioactive agent found in the essential oil of mountain celery seeds.

The unusual hypolipidemic activity of the methanolic fractionate of the essential oil (EOM) obtained from the mountain celery seed was previously reported. The most enriched 9(Z)-octadecenamide (oleamide) was speculated to be responsible for the relevant bioactivity. Chemically syntheized oleamide (CSO) yielded 85.1% with a purity of 98.6% when identified by RP-HPLC, FTIR, HREIMS, (1)H NMR, and (13)C NMR. CSO was tested for its antioxidative and hypolipidemic bioactivities. Results indicated CSO was potently hypolipidemic with regard to serum TG, TC, LDL-C, LDL-C/HDL-C, and hepatic TG (p < 0.05), but not for serum HDL-C and hepatic TC. In addition, CSO exhibited only poor antioxidative activity, implicating the possibility that the hypolipidemic and antioxidative bioactivity of original EOM was due to another coexisting constituent, probably gamma-selinene. Conclusively, oleamide is a potent hypolipidemic agent as regarding its effects on decreasing serum TG, TC, LDL-C and hepatic TG.

A novel biocompatible adhesive incorporating plant-derived monomers.

We describe a new class of biomaterials with potential for a variety of applications in tissue engineering, wound healing, and transdermal drug delivery. These materials are based on oleic methyl ester (OME), which is derived from various plant oils including soybean oil. The OME was acrylated (AOME) and subsequently copolymerized with methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) to form pressure sensitive adhesives (PSAs). We assessed the cytocompatibility of each PSA product using Alamar Blue and Live/Dead assays. It was found that after 2 h, human fibroblast cells attached on all four of the PSA polymers tested. After 24 h, cell spreading was seen on all materials with the exception of the polymerized AOME product (PAOME). Cells attached to the copolymer PSA products continued to proliferate for up to 2 weeks, as shown by fluorescent confocal microscopy imaging. Finally, a mechanical analysis of each of the copolymers is presented demonstrating that they have a range of mechanical properties and cell adhesiveness depending on the formulation, making them attractive candidates for use as bioactive adhesives.

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.

Analysis of chemically synthesized oleoylethanolamide by gas-liquid chromatography.

Oleoylethanolamide (OEA) is known to potentially have beneficial biological effects on weight management by controlling food intake and activating lipid catabolism. In biological fluids, OEA and other endogenously biosynthesized fatty acid ethanolamides are usually analyzed by liquid chromatography-mass spectrometry (LC-MS). The present study provides analytical method to routinely assess the quality of OEA prepared for biological studies by gas-liquid chromatography (GLC). The preparation of OEA for biomedical studies can be performed by N-acylation of oleic acid/esters or using oleoyl chloride. In the present study, OEA was prepared by transamidation of triolein. The analysis of the synthesized OEA has been performed by gas-liquid chromatography of its trimethylsilyl ether (TMS) derivatives. Free OEA cannot be analyzed as such because dehydration of the ethanolamide moiety promptly happens in the GLC injection. This thermal degradation reaction gives rise to the formation of an oxazoline derivative. The TMS moiety prevents the reaction, and the structure of the formed derivative was assessed by mass spectrometry. We show here that OEA prepared for biological studies can be routinely analyzed by GLC after TMS derivative preparation.

Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26.

We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes; however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs.

Novel sulfamide analogs of oleoylethanolamide showing in vivo satiety inducing actions and PPARalpha activation.

Long chain saturated and unsaturated alkyl sulfamide and propyl sulfamide derivatives, analogs of oleoylethanolamide, have been synthesized and evaluated in vivo and in vitro as peroxisome proliferator activated receptor alpha (PPARalpha) activators. Additionally, the anorexic effects of the new compounds have been studied in vivo in food-deprived rats. Among the active compounds N-octadecyl-N'-propylsulfamide (7) has been identified as a potent hypolipidemic compound, a potent feeding suppressant, and a concentration-dependent activator of PPARalpha.

In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide.

Oleamide (cis-9-octadecenamide) is a member of an emerging class of lipid-signaling molecules, the primary fatty acid amides. A growing body of evidence indicates that oleamide mediates fundamental neurochemical processes including sleep, thermoregulation, and nociception. Nevertheless, the mechanism for oleamide biosynthesis remains unknown. The leading hypothesis holds that oleamide is synthesized from oleoylglycine via the actions of the peptide amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The present study investigated this hypothesis using pharmacologic treatments, physiologic assessments, and measurements of serum oleamide levels using a newly developed enzyme-linked immunosorbant assay (ELISA). Oleamide and oleoylglycine both induced profound hypothermia and decreased locomotion, over equivalent dose ranges and time courses, whereas, closely related compounds, stearamide and oleic acid, were essentially without effect. While the biologic actions of oleamide and oleoylglycine were equivalent, the two compounds differed dramatically with respect to their effects on serum levels of oleamide. Oleamide administration (80mg/kg) elevated blood-borne oleamide by eight-fold, whereas, the same dose of oleoylglycine had no effect on circulating oleamide levels. In addition, pretreatment with the established PAM inhibitor, disulfiram, produced modest reductions in the hypothermic responses to both oleoylglycine and oleamide, suggesting that the effects of disulfiram were not mediated through inhibition of PAM and a resulting decrease in the formation of oleamide from oleoylglycine. Collectively, these findings raise the possibilities that: (1) oleoylglycine possesses biologic activity that is independent of its conversion to oleamide and (2) the increased availability of oleoylglycine as a potential substrate does not drive the biosynthesis of oleamide.

Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties.

Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-alpha (PPAR-alpha). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-alpha with high potency in vitro, and persistently reduce feeding when administered in vivo either parenterally or orally. The most potent of these compounds, (Z)-(R)-9-octadecenamide,N-(2-hydroxyethyl,1-methyl) (KDS-5104), stimulates transcriptional activity of PPAR-alpha with a half-maximal effective concentration (EC50) of 100 +/- 21 nM (n = 11). Parenteral administration of KDS-5104 in rats produces persistent dose-dependent prolongation of feeding latency and postmeal interval (half-maximal effective dose, ED50 = 2.4 +/- 1.8 mg kg(-1) i.p.; n = 18), as well as increased and protracted tissue exposure compared with OEA. Oral administration of the compound also results in a significant tissue exposure and reduction of food intake in free-feeding rats. These results suggest that the endogenous high-affinity PPAR-alpha agonist OEA may provide a scaffold for the discovery of novel orally active PPAR-alpha ligands.

Anticancer activity and possible mode of action of 4-O-podophyllotoxinyl 12-hydroxyl-octadec-Z-9-enoate.

4-O-Podophyllotoxinyl 12-hydroxyl-octadec-Z-9-enoate (PHEFE) is a structurally novel FA analog of podophyllotoxin. In the present study, in vitro effects of PHEFE on a panel of 60 human tumor cell lines and its potential modes of anticancer action were investigated. PHEFE exhibited strong growth-inhibitory action in a number of solid tumor cells in vitro. It did not inhibit tubulin polymerization as podophyllotoxin does; rather, it inhibited the catalytic activity of topoisomerase II. Flow cytometry and staining assay with 4,6-diamidine-2-phenylindole dihydrochloride showed that PHEFE blocked the cell cycle at the G2/M phase and induced apoptosis in HL-60 cells. These analyses suggest that PHEFE has promising anticancer characteristics that differ from podophyllotoxin and etoposide.

Ethyl oleate synthesis using Candida rugosa lipase in a solvent-free system. Role of hydrophobic interactions.

The solvent-free esterification reaction of a commercial oleic acid and ethanol was selected as the test reaction for Candida rugosa lipase immobilized on polypropylene (PP) at 318 K (initial molar ratio 1:1). Adding of water from 0 to 30 wt. % (in gram per gram of fatty acid x 100) and the pretreatment of Candida rugosa lipase with polyethylenglycol (PEG), octane, and acetone increases the conversion to ethyl esters. The role of hydrophobic interactions of the lipase with PP and PEG was studied using molecular mechanics (MM2) for calculation of steric energies and the parametrized model (PM3) for calculation of enthalpy changes upon interaction. The nonpolar lateral groups of amino acids interact strongly with PP, whereas polar groups interact more strongly with PEG. Both interactions stabilize the open, active conformation of the lipase from Candida rugosa. Activities ranged from 5 x 10(-5) to 2.0 x 10(-4) mol ethyl oleate/h/mg enzyme, depending on reaction conditions. Steric energy changes vary between +30 and -10 kcal/mol, whereas the enthalpy changes ranged from +10 to -10 kcal/mol.

A derivative of oleamide potently inhibits the spontaneous metastasis of mouse melanoma BL6 cells.

We reported previously that the abnormally augmented expression of connexin 26 (Cx26) is responsible for the enhanced spontaneous metastasis of mouse BL6 melanoma cells, and that the exogenous expression of a dominant negative form of Cx26 inhibits the spontaneous metastasis of BL6. Here we show that daily intraperitoneal (i.p.) injections of oleamide, a sleep-inducing lipid hormone, weakly inhibited the spontaneous metastasis of BL6 cells. To obtain a more effective reagent, 19 oleamide derivatives were chemically synthesized and tested for their ability to inhibit the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of Cx26 or Cx43. One of these, denoted metastasis inhibitor-18 (MI-18), inhibited the GJIC formed by Cx26 as well as oleamide but unlike oleamide, which is a non-selective inhibitor of connexin, it did not inhibit the GJIC formed by Cx43. Daily i.p. injections of MI-18 potently blocked the spontaneous metastasis of BL6 cells down to 15% of that in the untreated control mice. MI-18 was safe because even after >7 weeks of daily injections, the survival rate of the mice was 93%. We propose that MI-18 may serve as a novel and clinically important prototype of a potent inhibitor of spontaneous metastasis.