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Potential mechanisms by which Peceol increases the gastrointestinal absorption of amphotericin B.

PURPOSE: The purpose of this study was to ascertain how the incorporation of AmpB into a glyceride-rich excipient Peceol significantly increased Amphotericin B's (AmpB) gastrointestinal absorption in white male Sprague-Dawley rats. Based on preliminary studies, our working hypothesis was that incorporation of AmpB into mixed micelles composed of Peceol would significantly enhance gastro-intestinal (GI) tract absorption by increasing lymphatic drug transport and decreasing P-glycoprotein (PGP)-mediated drug efflux. METHODS: I. Lymphatic Transport STUDIES: Following an overnight fast (12-16 hr) and 48 hr postsurgery, rats were divided into two treatment groups and received a single-dose oral gavage (1 mL total volume) at 0700 h of either desoxycholate (DOC)-AmpB (5 mg AmpB/kg; n = 6 at each time point) or AmpB incorporated into 100% Peceol (Peceol-AmpB; 5 mg AmpB/kg; n = 6 at each time point). Mesenteric lymph samples were obtained prior to and at 0-4-hr, 4-6-hr, and 6-8-hr intervals post oral gavage. An equal volume of normal saline (1 mL) was administered intravenously to the animal following each blood draw to prevent fluid depletion throughout the duration of the study. Lymph was immediately harvested by centrifugation and analyzed for drug by high-performance liquid chromatography (HPLC). II. Multidrug Resistance 1 (mdr-1) STUDIES: Caco-2 cells were seeded at 10,000 cells/cm2 in T-75 flasks. When the cells reached 80% confluency, they were treated for 1 day and 7 days with 0.1% to 1.0% (v/v) Peceol or media alone (control). Following treatment, total RNA was isolated using TRIzol reagent, followed by reverse transcription into single-stranded cDNA. Polymerase chain reactions (PCR) were performed with specific primers for mdr-1. The PGP protein was determined by Western Blot Analysis. RESULTS: Mean weight of rats was not significantly different prior to and following drug administration. Similarly, kidney, liver, lung, spleen, and heart weights were not different between DOC-AmpB and Peceol-AmpB treatment group. A significantly greater amount of AmpB was transported through the mesenteric lymph duct for all the time intervals used following the administration of Peceol-AmpB treatment group compared to the administration of DOC-AmpB (suspension). A significant lower mdr-1 mRNA and PGP protein expression within Caco-2 cells was observed following 1 and 7 days treatment with Peceol 0.1% to 1.0% (v/v) compared to nontreated controls. CONCLUSIONS: Taken together, these findings suggest that Peceol increases the gastrointestinal absorption of AmpB by increasing the amount of drug that is transported through the mesenteric lymph duct and by decreasing mdr-1 mRNA and PGP protein expression, resulting in lower PGP-mediated AmpB efflux.

Administration, Oral↗

Triptolide loaded solid lipid nanoparticle hydrogel for topical application.

Triptolide (TP) has been shown to have anti-inflammatory, antifertility, antineoplastic, and immunosuppressive activity. However, its clinical usage is limited to some extent due to its poor water solubility and toxicity. In order to use innovative ways to administer TP and to overcome or alleviate its disadvantages, controlled-release delivery systems such as solid lipid nanoparticle(SLN(s)) have been developed. In the present paper we describe the preparation and some characterization of specialized delivery systems for TP. The transdermal delivery and anti-inflammatory activity were also evaluated. The results indicated that SLN could serve as an efficient promoter of TP penetrating into skin. Furthermore, different formulations were optimized in this study. The best formulation of SLN, consisted of tristearin glyceride, soybean lecithin, and PEG400MS, with a particle size of 123+/-0.9 nm, polydispersity index (PI) of 0.19, and zeta potential of -45 mV. When this SLN dispersion was incorporated into hydrogel, the nanoparticulate structure was maintained, and aggregation and gel phenomena of the particle could be avoided. The cumulative transdermal absorption rate in 12 h was 73.5%, whereas the conventional TP hydrogel was 45.3%. The anti-inflammatory effect is over two-fold higher than that of conventional TP hydrogel. Moreover, this SLN hydrogel consists of pharmaceutically acceptable ingredients, such as soybean lecithin and lipid, and the nanoparticle can improve safety and minimize the toxicity induced by TP.

Absorption↗

Enhanced drug dissolution and bulk properties of solid dispersions granulated with a surface adsorbent.

A combination of solid dispersion and surface adsorption techniques was used to enhance the dissolution of a poorly water-soluble drug, BAY 12-9566. In addition to dissolution enhancement, this method allows compression of the granulated dispersion into tablets. Gelucire 50/13 (polyglycolized glycerides) was used as the solid dispersion carrier. Hot-melt granulation was performed to adsorb the melt of the drug and Gelucire 50/13 onto the surface of Neusilin US2 (magnesium alumino silicate), the surface adsorbent. Dispersion granules using various ratios of drug-Gelucire 50/13-Neusilin US2 were thus prepared. The dissolution profiles of BAY 12-9566 from the dispersion granules and corresponding physical mixtures were evaluated using USP Type II apparatus at 75 rpm. The dissolution medium consisted of 0.1 N hydrochloric acid (HCl) with 1% w/v sodium lauryl sulfate (SLS). Dissolution of BAY 12-9566 from the dispersion granules was enhanced compared to the physical mixture. The dissolution of BAY 12-9566 increased as a function of increased Gelucire 50/13 and Neusilin US2 loading and decreased with increased drug loading. In contrast to the usually observed decrease in dissolution on storage, an enhancement in dissolution was observed for the dispersion granules stored at 40 degrees C/75% relative humidity (RH) for 2 and 4 weeks. Additionally, the flow and compressibility properties of dispersion granules were improved significantly when compared to the drug alone or the corresponding physical mixture. The ternary dispersion granules were compressed easily into tablets with up to 30% w/w drug loading. The extent of dissolution of drug from these tablets was greater than that from the uncompressed dispersion granules.

Adsorption↗

Formation of oligonucleotide adducts in pharmaceutical formulations.

During preformulation studies, we observed that oligonucleotide extracted from topical formulations contained considerable amounts of covalently modified oligonucleotide adducts. In this report, we describe the identification and characterization of reaction products that form when PS-oligodeoxyribonucleotide ISIS 2302 (1) is brought into contact with aqueous solutions of glycerol-derived excipients. Compatibility tests showed that the presence of certain glycerides in the formulation lead to adduct formation (1+58x amu, 1+72x amu, 1+58x+72y amu, x, and y are the number of modifications on one oligonucleotide strand). No adduct formation was observed in the presence of triglycerides or propylene glycol-derived excipients used in the study. Using nucleosides as model compounds, two modifications of deoxyguanosine were isolated by preparative reversed phase (RP)-high pressure liquid chromatography (HPLC) and characterized by nuclear magnetic resonance (NMR) and HPLC-mass spectrometry (MS). Modifications were identified as N2-(1-carboxymethyl)- and N2-(1-carboxyethyl) derivatives of 2'-deoxyguanosine. The mechanism of formation of these adducts may involve advanced glycation reactions possibly caused by excipient impurities or degradation products such as glyceraldehyde or glyceraldehyde derivatives.

Chemistry, Pharmaceutical↗

Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration.

The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.

Animals↗

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi).

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.

Autoradiography↗

Lipids of Acanthamoeba castellanii. Composition and effects of phagocytosis on incorporation of radioactive precursors.

The lipids of Acanthamoeba castellanii (Neff) consist of 52% neutral lipids and 48% polar lipids. Triglycerides account for 75% and free sterols for 17% of the neutral lipids. The major phospholipids are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), a phosphoinositide (6%), and diphosphatidylglycerol (4%). The phosphoinositide is unique in that it contains fatty acids, aldehyde, inositol, and phosphate in the ratio of 1.4:0.5:1.1, but it contains no glycerol. Sphingomyelin, cerebrosides, psychosine, and glycoglycerides were not detected, but small amounts of unidentified long chain bases and sugars are present. The rates of uptake of palmitate-1-(14)C and of its incorporation into glycerides and phospholipids were not affected by the phagocytosis of polystyrene latex beads. Although phagocytosis usually decreased the uptake by amebas of phosphate-(32)P, serine-U-(14)C, and inositol-2-(3)H, their subsequent incroporation into phospholipids was not demonstrably stimulated or inhibited by phagocytosis. Phagocytosis did seem to increase the incorporation into ameba phospholipids of phosphatidylcholine-1 ,2-(14)C but not that of phosphatidylethanolamine-1 ,2-(14)C. These experiments, in which the incorporation of radioactive precursors into total cell lipids was measured, do not, of course, eliminate the possibility that localized effects may occur.

Aldehydes↗

Nafenopin-induced hepatic microbody (peroxisome) proliferation and catalase synthesis in rats and mice. Absence of sex difference in response.

Nafenopin (2-methyl-2[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]-propionic acid; Su-13437), a potent hypolipidemic compound, was administered in varying concentrations in ground Purina Chow to male and female rats, wild type (Cs(a) strain) mice and acatalasemic (Cs(b) strain) mice to determine the hepatic microbody proliferative and catalase-inducing effects. In all groups of animals, administration of nafenopin at dietary levels of 0.125% and 0.25% produced a significant and sustained increase in the number of peroxisomes. The hepatic microbody proliferation in both male and female rats and wild type Cs(a) strain mice treated with nafenopin was of the same magnitude and was associated with a two-fold increase in catalase activity and in the concentration of catalase protein. The increase in microbody population in acatalasemic mice, although not accompanied by increase in catalase activity, was associated with a twofold increase in the amount of catalase protein. The absence of sex difference in microbody proliferative response in nafenopin-treated rats and wild type mice is of particular significance, since ethyl-alpha-p-chlorophenoxyisobutyrate (CPIB)-induced microbody proliferation and increase in catalase activity occurred only in males. Nafenopin can, therefore, be used as an inducer of microbody proliferation and of catalase synthesis in both sexes of rats and mice. The serum glycerol-glycerides were markedly lowered in all the animals given nafenopin, which paralleled the increase in liver catalase. All the above effects of nafenopin were fully reversed when the drug was withdrawn from the diet of male rats. During reversal, several microbody nucleoids were seen free in the hyaloplasm or in the dilated endoplasmic reticulum channels resulting from a rapid reduction in microbody matrix proteins after the withdrawal of nafenopin from the diet. Because of microbody proliferation and catalase induction with increasing number of hypolipidemic compounds, additional studies are necessary to determine the interrelationships of microbody proliferation, catalase induction, and hypolipidemia.

Animals↗

A novel and effective intranasal immunization strategy for respiratory syncytial virus.

In designing subunit vaccination strategies for respiratory syncytial virus (RSV), immunization by mucosal routes may present a realistic alternative to parenteral administration for inducing protective immune responses. To this end, we have utilized the BALB/c mouse model and an adjuvant formulation containing caprylic/capric glycerides (CCG) and polyoxyethylene-20-sorbitan monolaurate (PS). The intranasal (i.n.) delivery of purified natural F protein (3 microg per vaccine) formulated with CCG-PS resulted in the generation of statistically heightened serum anti-F protein immunoglobulin G (IgG), IgG1, IgG2b, and IgA antibodies. In addition, the presence of locally produced anti-F protein IgA was demonstrated in both vaginal and nasal washes of vaccinated mice. That production of specific serum and mucosal immunoglobulins resulted in functional immune responses was shown in neutralizing antibody assays and protection of mouse lungs against subsequent live virus challenge. Consequently, we propose a novel vaccine formulation composed of purified natural RSV F protein in CCG-PS as a viable intranasal immunogen to stimulate anti-RSV immune responses in humans.

Adjuvants, Immunologic↗

Effect of palm oil on lipid and lipoprotein metabolism and eicosanoid production in rats.

Palm oil (PA), which is very rich in palmitic acid, influenced the serum cholesterol concentration and tissue eicosanoid profile of rats characteristically. It increased the serum cholesterol concentration compared with safflower oil (SA), but the degree of the elevation was moderate compared with that of olive oil (OL). The proportion of arachidonic acid in tissue lipids was by no means lower on a PA diet than on an SA diet. The ratio of prostacyclin produced by the aorta to thromboxane A2 in plasma was not simply predicted by the dietary level of saturated fatty acid. PA tended to facilitate the utilization of arachidonate for prostacyclin in vitro in peritoneal macrophages compared with SA. Fatty acid profiles of PA rather than the glyceride structure or the tocotrienol appeared to be the major determinant for the specific features of lipid metabolism observed in rats fed PA.

Animals↗

Stearic acid metabolism and atherogenesis: history.

Studies conducted in dogs, rats, and hamsters show that stearic acid or stearic acid-rich glycerides are absorbed less efficiently than are lauric, myristic, and palmitic acids or their triglycerides. This observation may explain in part why stearic acid is less cholesterolemic than saturated fatty acids of shorter chain length. In rabbits, cocoa butter or other fats rich in stearic acid are less atherogenic than other saturated fatty acids. This finding is true for both cholesterol-containing and cholesterol-free diets.

Absorption↗

Effect on plasma lipids of interesterifying a mix of edible oils.

The aim was to establish whether interesterification of oils, an alternative to the use of trans fatty acids in margarine manufacture, adversely affects plasma lipids. Twenty-seven hypercholesterolemic men participated in a double-blind, crossover trial of three margarines: 1) high-linoleic acid, moderate trans fatty acids; 2) high-palm oil blend (predominantly lauric, myristic, palmitic, oleic, and linoleic acids); and 3) an interesterified form of the high-palm oil margarine. Both high-palm oil margarines led to similar low-density-lipoprotein (LDL) cholesterol concentrations (4.43 +/- 0.94 and 4.54 +/- 0.88 mmol/L, respectively), which were significantly higher than the LDL concentrations after the high-linoleic acid margarine (4.02 +/- 0.85 mmol/L, P < 0.001). Interesterification transferred substantial proportions of palmitic acid into the sn-2 position of glycerides and unsaturated fatty acids into the sn-1,3 positions, and this was reflected in plasma chylomicrons. This study shows that interesterification of oils used to harden margarines does not raise plasma cholesterol more than does the margarine's constituent fatty acids.

Cholesterol, HDL↗

Analysis of mixed lipid extracts using 1H NMR spectra.

A method has been developed for rapid analysis of lipid proton NMR spectra. Identification of lipid content is possible because of the presence of unique peaks or ratios of peaks for individual lipids. Spectra can be subdivided into regions where peaks represent certain chemical groups held in common, or uniquely by the various lipids. Vectors (B) are made up of the areas of these subdivisions of peaks from spectra of unknown components. A new FORTRAN algorithm, LIPICK, tests for the presence of unique peaks or combinations of peaks to determine which lipids may be present. The spectra vectors of known identified lipids are then placed in the (A) matrix of possible solution candidates. Quantitation of lipids in an 1H NMR spectrum (B) of an unknown mixture then proceeds by solving the equation AX = B for X (the concentrations of the individual lipids present) by singular value analysis. At this time, it is possible to test 1 mg of total lipid for the presence and relative concentration of 15 common lipids: cholesterol and its esters; phosphatidyl-ethanolamine, -choline, -serine, -inositol, -glycerol; tri- or di-glycerides; fatty acid; lysophosphatidylcholine; sphingomyelin; cerebrosides and sulfatides; dolichol and dolichol P; and phosphatidic acid. This procedure is suitable for membrane lipid analysis and has been evaluated using known mixtures of purified standard lipids.

Algorithms↗

The suppository form of antibiotic administration: pharmacokinetics and clinical application.

The rectal route of antibiotic administration might be used effectively when other routes of administration are inadequate or unsuitable. With the use of various adjuvants, the rectal route can provide satisfactory pharmacokinetics and acceptable local tolerance. Experiments in animals have demonstrated the influence of the pharmaceutical formulation of suppositories on the rectal absorption and systemic distribution of beta-lactams and aminoglycosides. In healthy volunteers and in children under treatment, similar adjuvants--mainly glyceride mixtures or non-ionic surface agents--have increased the rectal absorption of aminopenicillins, cephalosporins and macrolides. Other antibiotics, including metronidazole and cotrimoxazole, have been investigated in respect of their potential rectal administration.

Animals↗

Effects of age, diet and lactation on lipogenesis in rat adipose, liver and mammary tissues.

Four diets varying in safflower oil content from zero to 20% were used in a study of interactions among diet and physiological state. Increasing fat in the diet did not alter food intakes but decreased digestibility coefficients. Increasing safflower oil intake did not alter milk fat content in lactating rats but increased relative amounts of unsaturated fatty acids in milk fat. In liver and perirenal adipose tissues from young male and non-lactating female rats, low fat diets increased rates of lipogenesis from glucose in vitro and specific activities of enzymes whose functions are closely associated with lipogenesis. This adaptive hyperlipogenic response was not evident or was less prominent in aged or lactating rats. In the case of lactating rats it appears that lactation produces a marked reduction in adipose lipogenesis when low fat diets are fed. Reduced glyceride glycerol synthesis in lactating as compared to non-lactating rat adipose coupled with reduced fatty acid synthesis and in increased lipolysis indicated a shift in adipose function in the direction of increased fat mobilization as would be supportive of lactation. Only minor diet effects upon mammary enzyme patterns and rates of in vitro lipogenesis were observed.

Adipose Tissue↗

Effect of retinoic acid and retinyl acetate feeding upon lipid metabolism in adrenalectomized rats.

Many reports have appeared in the literature suggesting that vitamin A may exert some of its effects via changes in adrenocortical activity. A series of experiments were performed in order to assess the possible role of the adrenal gland in vitamin A-induced lipid alterations in rats. Adrenalectomized, sham-operated, and intact rats were fed retinoic acid or retinyl acetate at several levels. Either 25 or 100 retinol equivalents (RE)/g dry diet were fed to male Sprague-Dawley rats for periods of 7 or 28 days. Neither compound had an effect on the concentration of liver glycerides, phospholipids, cholesterol, or total lipids. Vitamin A, especially in the form of retinoic acid, was found to induce an elevation of plasma triglycerides. The presence of the adrenal gland was not necessary for the induction of hypertriglyceridemia nor was there any indication of increased adrenocortical output (as measured by plasma corticosterone level) as a result of vitamin A feeding. There was a reduction in circulating retinol as a result of retinoic acid feeding at either 25 or 100 RE in sham-operated and adrenalectomized rats but not in unoperated rats. These experiments demonstrate that vitamin A, especially in the form of retinoic acid, fed at as low as 25 RE/g diet to the rat can induce hypertriglyceridemia, and that the adrenal gland does not mediate this effect.

Adrenal Glands↗

Effects of dietary essential fatty acid concentration upon prostanoid synthesis in rats.

The effects of dietary linoleate as zero to 27% of energy, fed to female rats for 6 months, in relation to ability of whole blood to synthesize PG during clotting at 37 degrees was studied. Synthesis of PGE1, PGE2, PGF2 apha and TXB2 after 10 or 40 minutes of incubation of whole blood was determined by assay of serum concentration by radioimmunoassay. Fatty acid composition of serum phospholipids, cholesteryl esters and acyl glycerides was determined. PG synthesis increased with linoleate as zero to 2% of kcal; from 2% up to 7% there was a decrease of PG synthesis; from 7% to 27% there was a linear increase. There was no pattern to PGF2 alpha maximum concentration. PGE2 had a linear increase from 0 to 30% linoleate. PGE1 decreased with 0 to 2% linoleate, then increased between 2 and 7% and maintained a plateau through 27%. The concentration of TXB2 was found to be about 20-40 times that of PGE2 and PGF2 alpha. The decline of PGE1 synthesis with 2-7% linoleate calories was correlated with 20:3n-6 concentration and PGE2 and PGF2 alpha production with 20:4. With dietary linoleate concentrations greater than 7.4% PG synthesis had a linear increase up to 27% of kilocalories, not correlated with fatty acids.

Animals↗

Influence of trans unsaturated fats on experimental atherosclerosis in rabbits.

To test the effects of trans unsaturated fatty acids (t-FA) on atherosclerosis, lipidemia and enzyme activities, rabbits were fed a semipurified, cholesterol-free diet containing 40% sucrose, 25% casein and 14% fat for 5 months. Two experimental diets provided either 6% (high) or 3.2% (low) t-FA. The control group was fed a fat of composition similar to the two experimental diets but free of t-FA. Serum cholesterol and triglycerides were elevated in the rabbits fed 6% t-FA. Liver glycerides were also elevated in this group. The fatty acids of plasma, erythrocytes, epididymal fat, liver microsomes and liver mitochondria reflected the dietary composition. Levels of aortic atherosclerosis were identical in the three groups. There were no significant differences in activity of five hepatic enzymes: glucose-6-phosphatase (microsomal), fatty acid synthetase (cytosolic), malate dehydrogenase, beta-hydroxybutyrate dehydrogenase and monoamine oxidase (mitochondrial).

Adipose Tissue↗