The metabolism and pharmacokinetics of phospho-sulindac (OXT-328) and the effect of difluoromethylornithine.

BACKGROUND AND PURPOSE: Phospho-sulindac (PS; OXT-328) prevents colon cancer in mice, especially when combined with difluoromethylornithine (DFMO). Here, we explored its metabolism and pharmacokinetics. EXPERIMENTAL APPROACH: PS metabolism was studied in cultured cells, liver microsomes and cytosol, intestinal microsomes and in mice. Pharmacokinetics and biodistribution of PS were studied in mice. KEY RESULTS: PS undergoes reduction and oxidation yielding PS sulphide and PS sulphone; is hydrolysed releasing sulindac, which generates sulindac sulphide (SSide) and sulindac sulphone (SSone), all of which are glucuronidated. Liver and intestinal microsomes metabolized PS extensively but cultured cells converted only 10% of it to PS sulphide and PS sulphone. In mice, oral PS is rapidly absorbed, metabolized and distributed to the blood and other tissues. PS survives only partially intact in blood; of its three major metabolites (sulindac, SSide and SSone), sulindac has the highest C(max) and SSone the highest t(1/2) ; their AUC(0-24h) are similar. Compared with conventional sulindac, PS generated more SSone but less SSide, which may contribute to the safety of PS. In the gastroduodenal wall of mice, 71% of PS was intact; sulindac, SSide and SSone together accounted for <30% of the total. This finding may explain the lack of gastrointestinal toxicity by PS. DFMO had no effect on PS metabolism but significantly reduced drug level in mouse plasma and other tissues. CONCLUSIONS AND IMPLICATIONS: Our findings establish the metabolism of PS define its pharmacokinetics and biodistribution, describe its interactions with DFMO and largely explain its gastrointestinal safety.

Phospho-ibuprofen (MDC-917) incorporated in nanocarriers: anti-cancer activity in vitro and in vivo.

BACKGROUND AND PURPOSE: Phospho-ibuprofen (P-I; MDC-917) inhibits the growth of colon cancer in mice. Here, we investigated the use of nanocarriers to improve its pharmacokinetics (PKs) and anti tumour efficacy. EXPERIMENTAL APPROACH: The cellular uptake and cytotoxicity of P-I encapsulated into liposomes and micelles, and its in vitro metabolic stability, were determined in cultures of human colon adenocarcinoma cells. The performance of liposomal P-I was further evaluated in PK studies in mice, and in a model of colon cancer xenografts in nude mice. KEY RESULTS: Liposomal P-I and micellar P-I showed significantly enhanced cellular uptake in the colon cancer cells. Liposomal P-I also demonstrated increased cytotoxicity in vitro. Free P-I was metabolized rapidly to ibuprofen in the presence of purified esterases. In contrast, liposomal P-I, and to a lesser extent micellar P-I, was resistant to esterase-mediated hydrolysis. In mice, liposomal P-I partially protected P-I from hydrolysis in the circulation, and improved the biodistribution of intact P-I and its metabolites compared to free P-I. Liposomal P-I was more effective at inhibiting the growth of human colon cancer xenografts in mice, which may be explained on the basis of its improved PK profile compared to free P-I. CONCLUSIONS AND IMPLICATIONS: Liposome encapsulation of P-I partially protected P-I from esterase-mediated hydrolysis in mice, enhanced the cytotoxicity and bioavailability of P-I and increased its efficacy at inhibiting the growth of human colon cancer xenografts. These results indicate that liposomes are suitable nanocarriers for the delivery of P-I, and that the anti-tumour potential of liposomal P-I merits further evaluation.

Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel.

PURPOSE: Paclitaxel is currently administered i.v. as a slow infusion of a solution of the drug in an ethanol:surfactant:saline admixture. However, poor solubilization and toxicity are associated with this drug therapy. Alternative drug delivery systems, including parenteral emulsions, are under development in recent years to reduce drug toxicity, improve efficacy and eliminate premedication. METHODS: Paclitaxel emulsions were prepared by high-shear homogenization. The particle size of the emulsions was measured by dynamic light scattering. Drug concentration was quantified by HPLC and in vitro drug release was monitored by membrane dialysis. The physical stability of emulsions was monitored by particle size changes in both the mean droplet diameter and 99% cumulative distribution. Paclitaxel potency and changes in the concentration of known degradants were used as chemical stability indicators. Single dose acute toxicity studies were conducted in healthy mice and efficacy studies in B 16 melanoma tumor-bearing mice. RESULTS: QW8184, a physically and chemically stable sub-micron oil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drug loading (8-10 mg/mL) having a mean droplet diameter of <100 nm and 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both in the presence and absence of human serum albumin. Based on single dose acute toxicity studies, QW8184 is well tolerated both in mice and rats with about a 3-fold increase in the maximum-tolerated-dose (MTD) over the current marketed drug formulation. Using the B16 mouse melanoma model, a significant improvement in drug efficacy was observed with QW8184 over Taxol. CONCLUSIONS: QW8184, a stable sub-micron o/w emulsion of paclitaxel has been developed that can be filter-sterilized and administered i.v. as a bolus dose. When compared to Taxol, this emulsion exhibited reduced toxicity and improved efficacy most likely due to the composition and dependent physicochemical characteristics of the emulsion.

Carvedilol-liposome interaction: evidence for strong association with the hydrophobic region of the lipid bilayers.

Carvedilol (Kredex, Coreg) is a multiple action antihypertensive drug that has been shown to protect cell membranes from lipid peroxidative damages. In this study the physical and structural effects of carvedilol on lipid bilayers are investigated by fluorescence techniques, differential scanning calorimetry and other physical methods. Carvedilol binds to liposomal membranes (9:1 DMPC:DMPG) strongly with an apparent binding constant on the order of 10(4) M-1 in PBS (pH 7.4). The characteristic changes in its intrinsic fluorescence properties when bound to liposomes suggest that this compound is situated in a non-polar environment. The Stern-Volmer and bimolecular quenching constants, determined using nitrate as the fluorescence quencher, for the free and bound carvedilol indicate that the carbazole moiety is at a depth of > 11 A in the lipid bilayer. Fluorescence anisotropy measurements show that, unlike the membrane probes DPH and TMA-DPH, carvedilol is relatively mobile, and does not have a rigidly-defined molecular orientation in the bilayers. Differential scanning calorimetry results indicate that carvedilol is an effective membrane "fluidizer' as it dose-dependently lowers the gel to liquid crystalline transition temperature and broadens the endothermic transition. Comparative studies of interactions of carbazole, 4-OH carbazole and carvedilol with the model liposomal membranes reveal a possible role of membrane-partitioning in their antioxidant efficacy. These findings are discussed in perspective with the membrane biophysical properties of different classes of therapeutic significant lipid antioxidants in mind.

Water-in-oil microemulsions containing medium-chain fatty acids/salts: formulation and intestinal absorption enhancement evaluation.

PURPOSE: Water-in-oil (w/o) microemulsions have been developed which, in addition to non-ionic medium-chain glycerides, incorporate ionic lipids, primarily medium-chain fatty acids, such as caprylic (C8) capric (C10) and lauric (C12) acids and their corresponding sodium salts. The absorption enhancing activity of w/o microemulsions incorporating these lipids was evaluated in the rat using Calcein (MW = 623) a water-soluble and poorly absorbed marker molecule. METHODS: Phase diagrams were constructed where C8/C10 or C12 fatty acids were treated as lipophilic surfactants and their sodium salts as hydrophilic ones. The anesthetised rat model was employed to evaluate Calcein absorption upon a single intraduodenal administration from a solution and the various w/o microemulsions. RESULTS: A wide range of clear and transparent w/o microemulsions were obtained at ambient temperature either in liquid or solid form when a fixed blend of medium chain fatty acid/salt was titrated by a fixed ratio of the oil containing the oil-soluble mono- and diglycerides and deionized water or physiological saline. Upon intraduodenal administration in the anesthetised rat, the absorption of Calcein was improved from about 2% in aqueous solution up to about 37% in w/o microemulsions. Solid and liquid formulations were equally effective in improving bioavailability. The absorption enhancement activity of the fatty acids/salts followed the order C8 approximately C10 > C12. Absorption enhancement of Calcein was significantly reduced in the absence or presence of low levels of C8/C10 mono-/diglycerides. CONCLUSIONS: These results further support the use of medium-chain glycerides and fatty acids/salts in microemulsion formulations to improve intestinal absorption of water-soluble compounds.

Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects.

PURPOSE: This review highlights the state-of-the-art in pharmaceutical microemulsions with emphasis on self-emulsifying systems, from both a physical and biopharmaceutical perspective. Although these systems have several pharmaceutical applications, this review is primarily focused on their potential for oral drug delivery and intestinal absorption improvement. METHODS: Physicochemical characteristics and formulation design based on drug solubility and membrane permeability are discussed. RESULTS: Case studies in which lipid microemulsions have successfully been used to improve drug solubilization/dissolution and/or intestinal absorption of poorly absorbed drugs/peptides are presented. CONCLUSIONS: Drug development issues such as commercial viability, mechanisms involved, range of applicability, safety, scale-up and manufacture are outlined, and future research and development efforts to address these issues are discussed.

Formulation and intestinal absorption enhancement evaluation of water-in-oil microemulsions incorporating medium-chain glycerides.

We developed self-emulsifying water-in-oil (w/o) microemulsions incorporating medium-chain glycerides and measured their conductance, viscosity, refractive index and particle size. Formulation of Calcein (a water-soluble marker molecule, MW = 623), or SK&F 106760 (a water-soluble RGD peptide, MW = 634) in a w/o microemulsion having a composition of Captex 355/Capmul MCM/Tween 80/Aqueous (65/22/10/3, % w/w), resulted in significant bioavailability enhancement in rats relative to their aqueous formulations. Upon intraduodenal administration the bioavailability was enhanced from 2% for Calcein in isotonic Tris, pH 7.4 to 45% in the microemulsion and from 0.5% for SK&F 106760 in physiological saline to 27% in the microemulsion formulation. The microemulsion did not induce gross changes in GI mucosa at a dosing volume of 3.3 ml/kg. These results suggest that water-in-oil microemulsion systems may be utilized for enhancement of intestinal drug absorption.

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies.

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.

Interaction of N-alkylanthracyclines with lipid bilayers: correlations between partition coefficients, lipid phase distributions and thermotropic behavior.

The thermotropic behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), or of DPPC in admixture with cardiolipin or cholesterol, in the presence of various N-alkyl derivatives of both adriamycin and adriamycin-14-valerate has been investigated by high sensitivity differential scanning calorimetry. The analogues, particularly the 14-valerate derivatives, which were most lipophilic as judged by their lipid/buffer, and to a lesser extent by their octanol/buffer, partition coefficients, were the most effective in depressing the tm of the investigated lipids; correlations, however, were not absolute. Other factors, such as the distribution of the drugs between the solid and liquid-crystalline phases of the bilayer, were also important to the observed membrane perturbations. With all anthracyclines, however, no major changes in the transition enthalpy were observed. In the case of vesicles prepared from pure DPPC, curve fitting analysis based on ideal solution theory (J.M. Sturtevant (1984) Proc. Natl. Acad. Sci. USA 81, 1398-1400) applied at relatively low drug concentrations where single peak transitions were produced, adequately described the differential scanning calorimetric results. At high drug concentrations, however, the presence of multi-peak transitions were indicative of non-ideality.

Sedimentation field flow fractionation of fused unilamellar vesicles: comparison with electron microscopy and gel filtration.

Sedimentation field flow fractionation (SF3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-A small unilamellar vesicles to 700-A large unilamellar vesicles (LUVs) and the slower conversion to 950-A LUVs at 4 degrees C was readily monitored by SF3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF3 are discussed and correlated with electron microscopy and gel filtration.

Micellization of fatty acyl-CoA mixtures and its relevance to the fatty acyl selectivity of acyltransferases.

The critical micelle concentrations (CMCs) of palmitoyl-CoA/stearoyl-CoA and palmitoyl-CoA/oleoyl-CoA mixtures in 0.050 M KPi, pH 7.4, a buffer used in enzymatic studies, were determined by fluorescence. Mixed micelle solution theory, analogous to the thermodynamic treatment of vapor pressure, was applied to calculate monomer and micelle compositions. The behavior of the palmitoyl-CoA/stearoyl-CoA mixture is ideal, while the palmitoyl-CoA/oleoyl-CoA mixture, although not exhibiting ideal behavior, can be fitted reasonably well by nonideal theory. In both mixtures, selective micellization takes place and, unlike the case of pure fatty acyl-CoAs, above the CMC of the mixtures the concentration of molecules free in solution is strongly dependent upon total concentration. The information derived from the present physical studies becomes important in enzymatic studies with membrane-bound acyltransferases, where selectivity toward various fatty acyl donors, presented as binary mixtures, is frequently observed.

Solubility of palmitoyl-coenzyme A in acyltransferase assay buffers containing magnesium ions.

The solubility of palmitoyl-CoA is strongly affected by Mg2+ concentrations commonly used in acyltransferase reactions. In 0.10 M Tris-HCl buffer at pH 7.4 or 8.5, all of the palmitoyl-CoA in 10 microM solutions and 90% of the palmitoyl-CoA in 100 microM solutions are precipitated by 1 mM Mg2+. In 0.05 M phosphate at pH 7.4, and in 0.10 M Tris-HCl containing 0.4 M KCl, the substrate remains soluble at Mg2+ concentrations below 4-5 mM. Above 5 mM Mg2+, palmitoyl-CoA is insoluble in all of these buffers. Substrate solubility could therefore be a limiting factor when free Mg2+ and fatty acyl-CoAs are present together during acyltransferase assays.

A scanning calorimetric study of the interaction of anthracyclines with neutral and acidic phospholipids alone and in binary mixtures.

High sensitivity differential scanning calorimetry was employed to study the thermotropic behavior of multilamellar vesicles of neutral and acidic phospholipids and binary mixtures thereof in the presence of anthracycline antibiotics. Adriamycin and its lipophilic analogue, N-trifluoroacetyladriamycin-14-valerate (AD32) were investigated and compared to chlorpromazine and quinidine with respect to their ability to affect the pretransition and the main transition of the phospholipids suspended in physiological buffer. With liposomes of neutral dipalmitoylphosphatidylcholine the observed effects paralleled to some extent the corresponding octanol/buffer partition coefficients, with adriamycin being the least effective. Calorimetric measurements on liposomes prepared from pure dipalmitoylphosphatidylglycerol or from binary mixtures of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine showed that modulation of bilayer properties by adriamycin was greatly enhanced in the presence of negatively charged lipid headgroups presumably as a result of electrostatic interactions. AD32 interacted differently from adriamycin with the acidic bilayers at low drug concentrations, in a manner similar to that of its interaction with neutral bilayers. At high drug concentrations both adriamycin and AD32 produced transitions with multiple peaks not exhibited by chlorpromazine and quinidine which may be the result of a specific association of the anthracyclines with dipalmitoylphosphatidylglycerol. All four drugs produced only minor changes in the enthalpy of the main transition of the investigated lipids. The present findings are discussed in terms of their possible physiological relevance.

Use of high-performance liquid chromatography to detect hydroxyl and superoxide radicals generated from mitomycin C.

Distinguishing between short-lived reactive oxygen species like hydroxyl and superoxide radicals is difficult; the most successful approaches employ electron spin resonance (ESR) spin-trapping techniques. Using the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO) to selectively trap various radicals in the presence and absence of ethanol, an HPLC system which is capable of separating the hydroxyl- and superoxide-generated DMPO adduct species has been developed. The radical-generated DMPO adducts were measured with an electrochemical detector attached to the HPLC system and confirmed by spin-trapping techniques. The HPLC separation was carried out on an ODS reverse-phase column with a pH 5.1 buffered 8.5% acetonitrile mobile phase. The advantage of the HPLC system described is that it permits the separation and detection of hydroxyl and superoxide radicals without requiring ESR instrumentation. The antineoplastic bioreductive alkylating agent mitomycin C, when activated by NADPH-cytochrome c reductase, was shown to generate both hydroxyl and superoxide radicals.

Physical properties of fatty acyl-CoA. Critical micelle concentrations and micellar size and shape.

Critical micelle concentrations (CMCs) of palmitoyl-CoA were determined by surface tension, conductivity, and fluorimetric measurements in a variety of buffers at several pH values and ionic strengths. They ranged from 7 to 250 microM and were frequently an order of magnitude higher than most reported values. The CMCs of stearoyl-CoA and oleoyl-CoA, determined fluorimetrically, were also high and consistent with the expected effects of chain length and unsaturation. The effects of ionic strength and temperature were analyzed to obtain the extent of counterion binding and the thermodynamic parameters of micellization. The values of delta H0, delta G0, and delta S0 obtained in 0.011 M Tris, pH 8.3, are -6 K X J X mol-1, -64 K X J X mol-1, and +193 J X mol-1 X K-1, and the average number of univalent ions bound per molecule in the micelles is 1.4. These values are within the range of those obtained for other univalent and polyvalent detergents. Analyzed by sedimentation and diffusion, the micelles are approximately spherical with an anhydrous mass of 50,000 daltons but with dimensions inconsistent with fully extended molecules. Correlation of the information obtained from the present physical studies with kinetic studies using long-chain fatty acyl-CoAs as enzyme substrates may be helpful for understanding the enzymology of these compounds, and some previously published kinetic studies of membrane-bound and soluble enzymes may bear reinterpretation.