Nontoxic lipid

Using PTLC six major fractions of lipids (phospholipids, free sterols, free fatty acids, triacylglycerols, methyl esters, and sterol esters) were separated from the skin lipids of chicken to smdy the penetration responses of Schistosoma cercaria and Austrobilharzia variglandis [79a]. To determine the structure of nontoxic lipids in lipopolysaccharides of Salmonella typhimurium, monophosphoryl lipids were separated from these lipids using PTLC. The separated fractions were used in FAB-MS to determine [3-hydroxymyristic acid, lauric acid, and 3-hydroxymyristic acids [79b]. 

When purified endotoxin was treated with 0.1 N HC1 for 30 min at 100 °C, the resulting nondialyzable residue was also free of KDO but was nontoxic (CELD50, >10 yg) and nonpyrogenic (FI401 20 yg) (Table IV). We have designated this product nontoxic lipid A (Detox) and demonstrated its low toxicity in four animal species (Table II). The nontoxic lipid A in combination with P3 and ACP (or CWS) retained a degree of tumor regressive potency (80% cures) similar to that observed with the toxic lipid A (88% cures) and the purified endoxtoxin (81% cures). 

Results of chemical analysis showed that the glucosamine and total fatty acid contents of the KDO depleted, toxic lipid A and the nontoxic lipid A were essentially the same but that the nontoxic lipid A was significantly lower in the phosphorus content (Table V). The molar ratio of glucosamine phosphorus fatty acids was 2 2 4 for the toxic lipid A and 2 1 4 for the nontoxic lipid A. The relative molar distribution of normal fatty acids (lauric, myristic, and palmitic acids) and the 3-hydroxymyristic acid did not indicate a correlation between the content of these components and toxicity. The nontoxic lipid A possessed as high a tumor regression activity when combined with CWS as did the purified... 

Table VI. Synergy Between Endotoxin (ET), Nontoxic Lipid A (Detox), ...

We showed the existence of a toxic and a nontoxic lipid A fraction in the acid hydrolyzed endotoxin preparation (23). These two fractions were separated and the composition was determined on the purified components so that we could relate specific structural features of lipid A to both toxicity and tumor regression activity (23). 

This nontoxic lipid may represent a potential candidate for use in the immunotherapy of human cancer. In addition, it will be interesting to determine to what extent this nontoxic lipid A can replace the toxic components in eliciting the numerous other biological activities of lipid A. 

Qureshi, N., Takayama, K., Ribi, E. Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium. J Biol Chem 257 (1982) 11808-11815. 

A presynthetic approach has also been used to prepare lipid-conjugated oligonucleotides (LONs) in order to improve cellular uptake and intracellular delivery of various ODNs. Thus, Grijalvo et al. [62] discovered lipid-siRNA with anti-inflammatory properties while Godeau et al. [63] synthesized nontoxic lipid-ON conjugates, which efficiently inhibit HCV internal ribosome entry site-dependent translation in human Huh7 cells. 

A proteinaceous particulate material has been described that is effective as an oil spill-dispersant composition [1450]. The material is a grain product (such as oats) from which lipids are removed through organic solvent extraction. When such compositions are applied to an oil spill, they will adsorb oil, emulsify it, and finally, disperse it. Moreover, the compositions are substantially nontoxic. 

Particles from cationic lipids may also be useful for antisense therapy of skin disease — a nontoxic increase in the oligonucleotide uptake by cultivated keratinocytes and a sebocyte cell line has been reported [66]. Moreover, cationic dendri-mers also efficiently transfer reporter gene DNA to human keratinocytes cultivated in vitro. In the skin of hairless mice, in vivo transfection was possible with complexes, yet reporter gene expression was localized to perifollicular areas. Transfection, however, failed with the naked plasmid. For prolonged contact, biodegradable membranes coated with dendrimer/DNA complexes were used [67]. This hints at a follicular uptake of these complexes and indicates that gene transfection also may be possible with human skin, which has a thicker stratum comeum compared with mouse skin (eight to ten vs. two to three layers [58]). 

Qureshi N, Takayama K, Kurtz R. (1991) Diphosphoryl lipid A obtained from the nontoxic hpopolysaccharide of Rhodopseudomonas Sphaeroides is an endotoxin antagonist in mice. Infect Immun 59 441 44. 

The ultrasound contrast agents are manufactured from nontoxic natural or synthetic biodegradable materials (e.g., lipids or proteins), and a small amount of an inert low-solubility non-reactive gas (e.g., perfluorocarbon). These components have been shown to be harmless to the patient unlike the tens of milliliters of concentrated viscous solutions of the widely used X-ray contrast agents (which may sometimes result in nephrotoxicity). 

Haloalkanes. Certain haloalkanes and haloalkane-containing mixtures have been demonstrated to potentiate carbon tetrachloride hepatotoxicity. Pretreatment of rats with trichloroethylene (TCE) enhanced carbon tetrachloride-induced hepatotoxicity, and a mixture of nontoxic doses of TCE and carbon tetrachloride elicited moderate to severe liver injury (Pessayre et al. 1982). The researchers believed that the interaction was mediated by TCE itself rather than its metabolites. TCE can also potentiate hepatic damage produced by low (10 ppm) concentrations of carbon tetrachloride in ethanol pretreated rats (Ikatsu and Nakajima 1992). Acetone was a more potent potentiator of carbon tetrachloride hepatotoxicity than was TCE, and acetone pretreatment also enhanced the hepatotoxic response of rats to a TCE-carbon tetrachloride mixture (Charbonneau et al. 1986). The potentiating action of acetone may involve not only increased metabolic activation of TCE and/or carbon tetrachloride, but also possible alteration of the integrity of organelle membranes. Carbon tetrachloride-induced liver necrosis and lipid peroxidation in the rat has been reported to be potentiated by 1,2- dichloroethane in an interaction that does not involve depletion of reduced liver glutathione, and that is prevented by vitamin E (Aragno et al. 1992). 

Use of Endotoxin in Cancer Immunotherapy and Characterization of Its Nontoxic but Active Lipid A Components... 

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

The results of the biological tests carried out on the purified monophosphoryl lipid A fractions are shown in Table XI. The chick embryo lethality test showed that both TLC-1 and -3 were nontoxic, whereas TLC-5 exhibited some toxicity. These results indicate that there might be two levels of toxicity based on structure. The presence or absence of the sugar 1-phosphate group (and possibly some other unknown group) would control the upper... 

Figure 3. Structure of nontoxic monophosphoryl lipid A. R, = 3-hydroxymyristoyl R2 = lauroyl, 3-myristoxymyristoyl, or H.

In conclusion, we have designed a synthetic vesicular DNA carrier that physically and functionally mimics an enveloped virus particle. To achieve an acceptable degree of encapsulation within the vesicle, we use a process that is essentially inverse to the preparation of cationic lipid-DNA complexes. A suitable DNA condensing agent is introduced that, at a certain critical concentration, conveys a weak net cationic charge to the condensed DNA that then interacts spontaneously with a liposome containing one or more anionic components. These DNA formulations behave distinctly different from classic cationic liposome DNA complexes in vitro in as much as they have been shown to be nontoxic, to display a traditional linear dose response, and to be serum-insensitive. 

To a large extent, therefore, the toxicities of esters tend to be those of their hydrolysis products. Two physical characteristics of many esters that affect their toxicities are relatively high volatility, which promotes exposure by the pulmonary route, and good solvent action, which affects penetration and tends to dissolve body lipids. Many volatile esters exhibit asphyxiant and narcotic action. As expected for compounds that occur naturally in foods, some esters are nontoxic (in reasonable doses). However, some of the synthetic esters, such as allyl acetate, have relatively high toxicities. As an example of a specific toxic effect, vinyl acetate acts as a skin defatting agent. 

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