In vitro influence of fatty acids and bilirubin on binding of mycophenolic acid to human serum albumin.

Mycophenolic acid (MPA) is 98-99% bound to albumin. Because MPA is restrictively cleared and has a low extraction coefficient, increase in its free fraction related to decreased albumin binding results in lower total concentrations but unchanged unbound concentrations. Multiple factors, including hypoalbuminemia, impaired renal function, and accumulated mycophenolic acid glucuronide are known to reduce MPA protein binding. Little is known about the influence of fatty acids and bilirubin on this issue. By using quenching fluorescence method, the aims of this study were to investigate in vitro the binding properties of MPA, then the influence of myristic acid and bilirubin on MPA binding to albumin. The estimate of dissociation constant (Kd) of MPA was 13.2 [CI 95 12.7-13.8] µM. In the presence of myristic acid (concentration range 4-100 µM), apparent Kd (Kd(app)) of MPA was approximately 1.5-10-fold greater. For myristic acid/albumin molar ratio reachable in clinical settings (2:1 and 5:1), Kd(app) of MPA rose about a factor 1.5 and 2.2, respectively. In the presence of bilirubin (concentration range 0.5-5 µM), Kd(app) of MPA was approximately 1.5-5-fold greater than MPA Kd. For bilirubin/albumin molar ratio reachable in clinical settings (1:4 and 1:2), Kd(app) of MPA rose about a factor 1.5 and 1.9, respectively. These data suggest that hypertriglyceridemia or cholestasis may significantly increase MPA free fraction in clinical settings, thereby lowering MPA total concentration in plasma while the free concentration remains unchanged. These results may help to optimize the therapeutic drug monitoring of MPA.

Effect of solvent on cytotoxicity and bioavailability of fatty acids.

Fatty acids (FAs) represent an important part of cell membranes and a source of energy. However, their abundance is linked with several diseases such as type 2 diabetes mellitus, and for this reason they are studied intensively. In this article we compare the two main methods of dissolving FAs for work in vitro, (i) dissolution in dimethylsulfoxide (DMSO) and (ii) conjugation with bovine serum albumin (BSA), and describe the effects of the solvent on cytotoxicity (determination of viability) and bioavailability (as shown by the impact on the gene expression of TNF-alpha). We have found that conjugation with BSA is significantly less cytotoxic than dissolution in DMSO and also yields greater bioavailability.

Absorption properties of micellar lipid metabolites into Caco2 cells.

To elucidate the absorption characteristics of dietary lipids in the human intestine, we investigated the cellular uptake of lipid metabolites using a differential monolayer of the Caco2 cells. As lipid metabolites, several free fatty acids and 2-monoacylglycerols, were formed a mixed micelle by bile salts and lysophospholipids and they were supplied to the Caco2 cells. To estimate the effect of the mixed micelles on the permeability of cells' membranes during incubation with the mixed micelles, the transepitherial electrical resistance (TEER) value was monitored, and no pronounced changes of TEER was detected. This suggested that mixed micelles did not affect their cellular properties of the barrier measured by TEER. The lipid metabolites transferred from the mixed micelle into the Caco2 cells were determined quantitatively by an enzymatic colorimetric method and were done by thin layer chromatography (TLC) for a species of acylglycerols. These highly sensitive methods enabled us to monitor the transepithelial transports of various kinds of non-isotope-labeled various lipid metabolites. Newly re-synthesized triacylglycerols were accumulated in Caco2 cells after 30 min incubation with the mixed micelles, and their amounts increased gradually for 4 h. The secretion of re-esterified triacylglycerols into a basolateral medium from the Caco2 cells began at 2 h after the mixed micelles were added to the apical medium. The intake of external lipid metabolites by the Caco2 cells were evaluated by an initial 2-h incubation with the mixed micelles. For example, 2-monomyristin and 2-monopalmitin were more rapidly transferred into the Caco2 cells from the mixed micelles than 2-monocaprin was. On the other hand, the absorption rates of capric acid, lauric acid and myristic acid by the cells were larger than those of stearic acid and oleic acid. It revealed that the side-chain structure of these lipid metabolites affected their absorption by the Caco2 cells. The results of this study suggested that the Caco2 cell monolayer could be a useful model for investigating the involvement of dietary lipids in the transepithelial absorption in the human intestine.

Safety assessment of myristic acid as a food ingredient.

Myristic acid is used in the food industry as a flavor ingredient. It is found widely distributed in fats throughout the plant and animal kingdom, including common human foodstuffs, such as nutmeg. Myristic acid (a 14-carbon, straight-chain saturated fatty acid) has been shown to have a low order of acute oral toxicity in rodents. It may be irritating in pure form to skin and eyes under exaggerated exposure conditions, but is not known or predicted to induce sensitization responses. Myristic acid did not induce a mutagenic response in either bacterial or mammalian systems in vitro. Relevant subchronic toxicity data are available on closely related fatty acid analogs. In particular, a NOEL of >6000mg/kg was reported for lauric acid (a 12-carbon, straight-chain saturated fatty acid) following dietary exposure to male rats for 18 weeks and a NOEL of >5000mg/kg was reported for palmitic acid (a 16-carbon, straight-chain saturated fatty acid) following dietary exposure to rats for 150 days. The data and information that are available indicate that at the current level of intake, food flavoring use of myristic acid does not pose a health risk to humans.

Pharmacokinetics and tissue distribution of zidovudine in rats following intravenous administration of zidovudine myristate loaded liposomes.

Liposomes accumulating in the reticuloendothelial system (RES) appear to be a promising vehicle to improve the therapeutic index of anti-HIV drugs such as zidovudine (AZT). Since the entrapment efficiency of AZT in liposomes was found to be low and AZT leakage from liposomes is fast, zidovudine myristate (AZT-M) was synthesized as a prodrug, and AZT-M incorporated liposomes in a lyophilized form were prepared with an average diameter of 90 nm and an encapsulation efficiency of 98% after reconstitution. The pharmacokinetic profiles and tissue distribution of AZT after i.v. administration of AZT-M liposomes in rats were investigated, and the results were compared with those after i.v. administration of AZT solution. AZT levels in plasma were significantly higher following application of AZT-M liposomes compared with AZT solution, and AUC0_infinity increased from 5.0 +/- 0.7 micromol x min x ml(-1) to 8.2 +/- 1.7 micromol x min x ml(-1) accordingly. Tissue distribution studies also confirmed higher concentrations of AZT in organs of RES and brain, suggesting that AZT-M liposomes might be promising candidates for therapy of HIV infections.

Synchrotron FT-IR microscopic study of chemical enhancers in transdermal drug delivery: example of fatty acids.

This article illustrates the analysis by synchrotron infrared microscopy of skin treated with penetration enhancers. Pig skin was treated with two fatty acids commonly employed as penetration enhancers, palmitic (C16) and myristic (C14) acids, in propylene glycol (PG). The use of perdeuterated fatty acid chains enabled the penetrating molecules to be perfectly distinguished from the endogenous lipids due to the difference between C-D and C-H stretching modes. Palmitic acid was detected in the stratum corneum (SC), a particularly alkyl-rich region, whereas myristic acid penetrates deeper into the epidermis. Similar experiments with lead and calcium soaps were also performed, but no detectable signal was observed, indicating a much weaker penetration. Additionally, the C-D2 stretching frequency provides information about the conformational order of the penetrating molecules inside the skin. The results indicate that fatty chains are in an ordered state. The improved spatial resolution allows the determination of both chemical composition and distribution in the different layers, from the SC to the dermis.

Permeation of a myristoylated dipeptide across the buccal mucosa: topological distribution and evaluation of tissue integrity.

The ex vivo permeation of an acylated model dipeptide, Myristoyl-Tryptophan-Leucine (Myr-Trp-Leu) was studied using pig buccal mucosa. Myr-Trp-Leu, being lipophilic, did not readily penetrate across the membrane. Rather, it accumulated in the epithelial and connective tissue of the mucosal barrier. The topological distribution of Myr-Trp-Leu across the mucosa, following its application in ethanol/phosphate buffer (30/70 pH 7.4), was determinated by thin-sectioning of the tissue, extraction of the peptide, and high performance thin layer chromatography (HPTLC). The concentration profile depended, of course, on the duration of the experiment and appeared to be dependent upon the presence of sufficient ethanol in order that the peptide could be solubilized. This important role for ethanol then raised the question of the solvent's effect on tissue integrity. Light microscopic examination of the mucosa was, therefore, undertaken, under identical conditions to those used in the permeation experiments, to evaluate any perturbation induced by the ethanolic vehicle. No obvious effects were observed.