Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

Bioactive lipids derived from the metabolism of polyunsaturated fatty acids are important mediators of the inflammatory response. Labor per se is considered a sterile inflammatory process. Intra-amniotic inflammation (IAI) due to microorganisms (i.e., intra-amniotic infection) or danger signals (i.e., sterile IAI) has been implicated in the pathogenesis of preterm labor and clinical chorioamnionitis at term. Early and accurate diagnosis of microbial invasion of the amniotic cavity (MIAC) requires analysis of amniotic fluid (AF). It is possible that IAI caused by microorganisms is associated with a stereotypic lipidomic profile, and that analysis of AF may help in the identification of patients with this condition. To test this hypothesis, we analyzed the fatty acyl lipidome of AF by liquid chromatography-mass spectrometry from patients in spontaneous labor at term and preterm gestations. We report that the AF concentrations of proinflammatory lipid mediators of the 5-lipoxygenase pathway are significantly higher in MIAC than in cases of sterile IAI. These results suggest that the concentrations of 5-lipoxygenase metabolites of arachidonic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4 in particular could serve as potential biomarkers of MIAC. This finding could have important implications for the rapid identification of patients who may benefit from anti-microbial treatment.-Maddipati, K. R., Romero, R., Chaiworapongsa ,T., Chaemsaithong, P., Zhou, S.-L., Xu, Z., Tarca, A. L., Kusanovic, J. P., Gomez, R., Chaiyasit, N., Honn, K. V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome.

Clinical chorioamnionitis at term (TCC) is the most common obstetrical infliction diagnosed in labor and delivery units worldwide and is associated with a substantial increase in maternal and neonatal morbidity and mortality. This obstetrical complication is a heterogeneous condition, as only half of patients have detectable microorganisms in the amniotic cavity. Because bioactive lipids play a key role in the initiation and resolution of an inflammatory response, we aimed to characterize the amniotic fluid lipidome in patients with TCC. We studied the amniotic fluid of patients in the following groups: 1) spontaneous labor at term without clinical chorioamnionitis (TLB) and 2) spontaneous labor at term with clinical chorioamnionitis (TCC). The TCC group was subdivided into a) those with microbial invasion of the amniotic cavity (TCC-MIAC) and b) those without microbial invasion of the amniotic cavity (TCC-noMIAC). The amniotic fluid concentration of proinflammatory lipid mediators did not differ between patients in TLB with TCC. In contrast, concentration of lipids with anti-inflammatory/proresolution properties was significantly lower in all patients with TCC than in those with TLB. These results suggest that while proinflammatory lipid mediators are involved in infection-driven intra-amniotic inflammation, a relative deficiency of anti-inflammatory/proresolution lipid mediator biosynthesis is a characteristic of TCC.

Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor.

Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography-mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition.

Stability and analysis of eicosanoids and docosanoids in tissue culture media.

Bioactive lipids derived from polyunsaturated fatty acids constitute an important set of lipid mediators with diverse homeostatic and pathophysiological activities. The role of bioactive lipids in inflammation, active resolution of inflammation, reproduction, cardiovascular biology, chronic diseases such as cancer and metabolic disorders is an active area of research. Other than the few clinically used eicosanoids such as prostaglandin F(2)α, prostacyclin, and prostaglandin E(2), little is known about the stability of a majority of the bioactive lipids in aqueous solutions despite their extensive use under in vitro experimental conditions. The present study evaluated the stability of eicosanoids and docosanoids as well as their parent polyunsaturated fatty acids in tissue culture media with respect to incubation time, temperature, and composition of the media using multiple reaction monitoring (MRM) LC-MS. The results provide a comprehensive and valuable data set on the stability of these bioactive lipid mediators to guide in vitro experimentation.

Bioavailability and pharmacokinetics of the investigational anticancer agent XK469 (NSC 698215) in rats following oral and intravenous administration.

PURPOSE: To determine the oral bioavailability of R-XK469, a water-soluble investigational anticancer agent undergoing phase I clinical trials as an intravenous product. METHODS: R-XK469 was administered to two groups of catheterized Sprague-Dawley rats via the oral and IV routes at a dose of 10 mg/kg and blood samples were collected at predetermined times. XK469 in plasma samples was quantified using a HPLC method. The pharmacokinetic parameters were computed using WinNonlin 4.0.1 software. RESULTS: The pharmacokinetic parameters of XK469 following oral and IV administrations, respectively, were (mean+/-SD): C(max) 138+/-64 and 404 +/- 355 microg/ml; AUC(0-infinity) 2381 +/- 773 and 2854 +/- 1924 microg h/ml; and elimination half-life (T(1/2)) 12.9 +/- 5.8 and 13.5 +/- 7.8 h T(max) was 2.92+/-1.92 h following oral dosing. Oral R-XK469 was 83% bioavailable. CONCLUSION: Together with the antitumor efficacy of oral XK469 shown in preclinical models and its schedule dependency, these results indicate the promise of developing an oral dosage form of R-XK469 for clinical development.

Pharmacokinetics and tissue distribution of cryptophycin 52 (C-52) epoxide and cryptophycin 55 (C-55) chlorohydrin in mice with subcutaneous tumors.

PURPOSE: To compare the pharmacokinetics and tissue distribution (both normal and tumor) of cryptophycin 52 (C-52) and its putative chlorohydrin prodrug cryptophycin 55 (C-55) in a murine model and to investigate a possible mechanism behind the superior activity of C-55. METHODS: Mammary adenocarcinoma 16/c tumor-bearing mice were treated with an i.v. bolus of 11 mg/kg C-52 or 38 mg/kg C-55 in Cremophor-alcohol. At predetermined time intervals, C-52 and C-55 concentrations in plasma, liver, kidney, small intestine and tumors were measured using a previously described HPLC method. Pharmacokinetic parameters were computed using noncompartmental methods. Tissue (both normal and tumor) to plasma ratios as a function of time were also calculated for comparison. RESULTS: Both C-52 and C-55 were rapidly distributed into different tissues including tumors following i.v. administration. However, the affinities of these compounds towards different tissues were different. Thus, the half-lives (minutes) of C-55 were in the decreasing order liver (725), intestine (494), tumor (206), kidney (62) and plasma (44), whereas the AUC values (microg x min/ml) were in the order tumor (9077), liver (7734), kidney (6790), plasma (2372) and intestine (2234). For C-52, the half-lives (minutes) were in the decreasing order liver (1333), kidney (718), intestine (389), tumor (181) and plasma (35), and the AUC values (microg x min/ml) were in the order kidney (1164), liver (609), intestine (487), plasma (457) and tumor (442). The relative exposures to C-52 after i.v. injection of C-55 were plasma 3.9%, tumor 80.8%, kidney 3.4%, liver 1.1% and intestine 2.8%. Although plasma exposure to C-52 following C-55 administration was relatively small, the use of C-55 to deliver C-52 increased the retention of C-52 and its AUC in tumor compared to direct injection of C-52. Simultaneously, this approach shortened C-52 retention in all normal tissues studied. CONCLUSIONS: The distribution of C-55 and its bioconversion to C-52 in different organs and tumor tissue observed in this study suggest the ability of C-55 to target tumor tissue, creating a depot of C-52 in tumor. Increased C-52 exposure of tumor, with concomitant decreased exposure of normal tissue, is a contributing factor to the superior activity of C-55 versus C-52. However, except in the case of tumor tissue in which 81% of C-55 converts to C-52, only a minor amount of C-55 may serve as a prodrug for C-52, whereas the majority is handled by the biosystem through a different route of elimination. Tissue distribution combined with rate of conversion may be an important determinant of the relative effectiveness of other epoxide-chlorohydrin pairs of cryptophycins.

Lecithin vesicles for topical delivery of diclofenac.

Skin penetration of topically applied diclofenac is important for the treatment of rheumatic diseases and actinic keratoses. We have studied the permeation of diclofenac across human cadaver epidermis in-vitro from four lecithin vesicle formulations and a few marketed semi-solid preparations. The lecithin vesicle formulations were prepared by dissolving the lipid contents (lecithin and sodium cholate) in a 1:1 mixture of methanol-chloroform, evaporating the solvents under vacuum, and hydrating the lipid layer with the drug solution in water or 10% ethanol. The vesicles were sonicated for 5 min to reduce the vesicle size and their size and Zeta potential were characterized. The cumulative amount and maximum flux of diclofenac was 69.7+/-40.3 micrograms and 4.77+/-3.16 micrograms/hcm(2) from lecithin vesicles containing sodium cholate and 10% ethanol, and is the highest of all formulations studied. The cumulative amount and mean maximum flux obtained from other formulations were in the range of 2.46+/-1.98-29.9+/-10.1 micrograms and 0.53+/-0.46-3.61+/-0.86 micrograms/hcm(2). Based on the results, lecithin vesicles of diclofenac appear to be advantageous for the topical delivery of diclofenac.

A novel method for determination of drug solubility in polymeric matrices.

Crystallization of drugs in metastable, supersaturated adhesive polymeric matrices in transdermal drug delivery devices can be avoided by determination of the solubility of the drug in the adhesive polymer. A novel method is described to determine the solubility of the drug in polymeric matrices. Unlike existing methods, this method does not require a long and uncertain experimental time, and is accurate. In this study, an easy and accurate method is presented for the determination of solubility of drugs in polymers based on the relationship between thermodynamic activity of drugs and steady-state flux. In particular, the steady-state flux from a reference saturated solution across a test membrane was compared to an experimentally determined relationship between the polymeric loading concentration and the observed steady-state fluxes. The validity of this method was demonstrated by comparing the results to microscopic observation of crystallization and the study of aged drug-loaded adhesives for lidocaine as a model drug and an acrylate pressure-sensitive adhesive as a model polymer. The solubility of lidocaine was 20.8 +/- 0.5% (w/w) in the acrylate polymer.

Iontophoresis of lecithin vesicles of cyclosporin A.

Site-specific immunosuppression with topical cyclosporin A (CsA) has broad clinical implications in the treatment of skin disorders like psoriasis, pyoderma gangrenosum, lichen planus, cutaneous graft-versus-host disease and contact hypersensitivity and the temporary treatment of skin allografts on burn wounds. However, like any other peptide drug, its skin delivery is hindered by the barrier property of stratum corneum and the physicochemical properties of CsA. We have attempted to deliver CsA across human cadaver epidermis in vitro using colloidal systems like microemulsion and lecithin vesicles and iontophoresis. Although, passive diffusion did not result in permeation of quantifiable amounts of CsA, anodal iontophoresis of the negatively charged colloidal systems facilitated the permeation. Electroosmosis and compromised epidermis might have contributed to the higher skin flux. Lecithin vesicles were better than microemulsion for the iontophoretic delivery of CsA and appear to have potential in site-specific immunosuppression.