Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phosphodiester ether lipid derivatives.

Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.

Progesterone freeze-dried systems in sublingual dosage form.

Various polymer matrices were tested to enhance progesterone bioavailability as part of an emergency therapy. Among the different polymers used, i.e. poly(N-vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), Dextran T70 and partially saponified poly(methyl glyoxylate) (PMGz), the latter gives the fastest solubilization rate. The best results were obtained with the lyophilized dosage form instead of a simple mixture of the drug within the polymer matrix. A nearly instantaneous solubilization was observed with PMGz copolymers bearing 10-40% of carboxylic groups and containing up to 20% of the drug. The instantaneous solubilization of the PMGz matrix is due to the hydrophilic moieties, and the presence of hydrophobic zones in PMGz promotes good affinity with the drug and optimal dispersion into the matrix.

The total syntheses of D-erythro-sphingosine, N-palmitoylsphingosine (ceramide), and glucosylceramide (cerebroside) via an azidosphingosine analog.

The total synthesis of D-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose via an azidosphingosine intermediate to give highly homogeneous (>99.9% C18:1) sphingosine base (9) which contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel method utilizing Mosher's acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous (99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for biophysical studies of the structures of their hydrated bio-molecular assemblies.

The total synthesis of ganglioside GM3.

Previous syntheses of ganglioside GM3 (NeuAc alpha3Gal beta4Glc beta1Cer) are reviewed, and both chemoenzymatic and chemical total synthetic approaches were investigated. In a chemoenzymatic approach, (2S,3R,4E)-5'''-acetyl-alpha-neuraminyl-(2''' --> 3'')-beta-galactopyranosyl-(1'' --> 4')-beta-glucopyranosyl-(1' <--> 1)-2-azido-4-octadecene-1,3-diol (azidoGM3) was readily prepared utilizing recombinant beta-Gal-(1'' --> 3'/4')-GlcNAc alpha-(2''' --> 3'')-sialyltransferase enzyme, and was evaluated as a synthetic intermediate to ganglioside GM3. The chemical total synthesis of ganglioside GM3 was performed on one of the largest scales yet reported. The highlights of this synthesis include minimizing the steps necessary to prepare the lactosyl acceptor as a useful anomeric mixture, which was present in excess for the highly regioselective and fairly stereoselective sialylation with a known neuraminyl donor to give the protected GM3 trisaccharide. The synthetic methodology maximized convergence by a subsequent glycosidic coupling of the well-characterized GM3 trisaccharide trichloroacetimidate derivative with protected ceramide. The ganglioside GM3 was nearly homogeneous as the two glycosidic couplings utilized preparative HLPC purifications, and variations in the sphingosine base and fatty acyl group were under 0.1 and 0.2%, respectively.

Unusual hydration properties of C16:0 sulfatide bilayer membranes.

After deacylation of bovine brain sulfatide under mild alkaline conditions and reacylation using palmitoyl chloride (, Chem. Phys. Lipids. 34:41-53), the anionic glycosphingolipid N-palmitoyl galactosulfatide (C16:0-GalSulf) has been synthesized. By differential scanning calorimetry (DSC), anhydrous C16:0-GalSulf exhibits an endothermic transition, T(M) = 93 degrees C (DeltaH = 5. 5 kcal/mol C16:0-GalSulf) on heating. With increasing hydration (50 mM sodium phosphate buffer, pH 7.0; 50 mM NaCl), T(M) decreases, reaching a limiting value of 49 degrees C (DeltaH = 8.2 kcal/mol C16:0-GalSulf) at 20 wt% buffer. X-ray diffraction data have been recorded over the hydration range 0-62% at temperatures below (20 degrees C) and above (60 degrees C) T(M). At 20 degrees C, sharp wide-angle reflections at approximately 1/4.4 A(-1), approximately 1/4.1 A(-1), and approximately 1/3.8 A(-1) indicate the presence of an ordered-chain gel phase, whereas at 60 degrees C a broad reflection at 1/4.5 A(-1) characteristic of a melted-chain phase is observed. Lamellar diffraction patterns consistent with the presence of bilayer phases are observed at both temperatures. At 60 degrees C, in the liquid-crystalline L(alpha) phase, the bilayer periodicity increases with hydration, in both water and 100 mM Na(+) buffer. Interestingly, in the gel phase at 20 degrees C, the bilayer periodicity (d = 64 A) is insensitive to hydration (over the range 30-60 wt%) with either water or buffer. The continuous swelling behavior exhibited by the L(alpha) bilayer phase of C16:0-GalSulf is typical of lipids bearing a net negative charge and confirms that the presence of 100 mM Na(+) is insufficient to shield the charge contributed by the sulfate group. In contrast, the lack of continuous swelling behavior of the bilayer gel phase of C16:0-GalSulf is unusual and resembles that of Na(+) soaps. Thus, presumably, alterations in the surface charge characteristics of the C16:0-GalSulf bilayer occur on hydrocarbon chain melting and lead to major changes in lipid hydration.

Stability assessment of ketoconazole in aqueous formulations.

Ketoconazole is an imidazole antifungal agent. It has a wide antifungal spectrum and possesses some antibacterial activity. In inappropriate formulations, especially in aqueous media, ketoconazole molecules may be unsteady. The stability of ketoconazole in aqueous media was assessed as a function of pH, antioxidant and ketoconazole concentrations. It was found that ketoconazole was least stable at pH 1 among the pH values studied (pH 1-9). Since the major degradation pathway was specific acid catalysis, based upon the transition-state theory, the entropy (DeltaS) of the activation was calculated and found to be negative indicating that the activated complex was more constrained than the individual species. The free energy of activation (DeltaG) was estimated to be 30 kcal mol(-1). The viscosity of the formulation was found to be more stable at high pH because carbopol is stable at basic pH and protected ketoconazole. It appears that the amount of ketoconazole in the formulation has a low influence on the degradation mechanisms. The increase of the butylated hydroxytoluene antioxidant levels from 0.05 to 0.4%, adversely affected the stability of ketoconazole. In conclusion, the expected shelf life of the final ketoconazole formulation (pH 7, 0.1% butylated hydroxytoluene) was 15 months.

Rheological behavior of drug suspensions in Gelucire mixtures and proxyphylline release from matrix hard gelatin capsules.

Mixtures of Gelucires 50/02 and 50/13 showing different hydrophilic-lipophilic balances (HLB) and of proxyphylline were used to prepare suspensions at a concentration of 25% and to manufacture extended release hard gelatin capsules by cooling. The rheological behaviors of Gelucire mixtures with and without drug were determined by adjustment of the rheograms to the Ostwald power-law and by statistical assessment of the flow index. Pure Gelucire mixtures were very slightly shear thickening whereas proxyphylline suspensions had a thixotropic shear thinning behavior. These rheological behaviors can be explained by the chemical composition and by the ratio of the two Gelucires used. Extended release of proxyphylline was obtained with all these mixtures. Drug release increased with Gelucire mixture HLB owing to higher erosion. A viscosity-release relationship was found and allowed, with these two Gelucires of extreme HLB and viscosities, to define the formulations which will give an optimal drug release, by the determination of their suspension viscosity. Modeling of dissolution kinetics has generally shown the predominance of surface erosion of the plugs relative to drug diffusion inside the matrix. This was confirmed by the better linearization of percentage released, according to Hixson-Crowell as compared with Higuchi.

Rheology of polyol behenates and drug release from matrix monolithic capsules.

Three polyol behenates with similar melting points (MP) and different hydrophilic-lipophilic balances (HLB) were studied (MP/HLB: 70/02, 63/05 and 57/13). After melting at MP+30 degrees C, the rheological behaviour of behenates was determined by adjustment of the rheograms to the Ostwald power-law and by statistical assessment of the flow index. Behenates showed slight shear thickening. This shear thickening increased when HLB of behenates decreased. This behaviour accounted for a reorganization of the particles under the shear, which became easier when the proportion of the polyethylene glycol chains in the wax decreased. Proxyphylline was used to prepare suspensions at a concentration of 25% in the melted behenates, and to manufacture monolithic capsules by cooling. The suspensions had a shear-thinning behaviour with or without thixotropy. Colloidal particles and aggregates formed in these suspensions directly influenced the rheological properties, as observation of solidified suspensions by scanning electron microscopy confirmed. Extended release of proxyphylline was obtained with the three waxes. Behenates 63/05 and 70/02 gave inert matrices and released drug very slowly. Hydrodispersible behenate 57/13 swelled and made up a kind of hydrophilic matrix that released proxyphylline more quickly, due to slight erosion. In the three cases, the release mechanism was basically diffusional in nature.

Structure and properties of totally synthetic galacto- and gluco-cerebrosides.

The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides.

Syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and analogs with 13C- and 2H-labeled choline head groups.

The syntheses of four head group labeled analogs of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) (6) by a general method from 1,2-di-O-palmitoyl-sn-glycero-3-phosphatidic acid (5) have been performed. The syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phospho[alpha-13C]choline (6a) and 1,2-di-O-palmitoyl-sn-glycero-3-phospho[beta-13C]choline (6b) were performed from labeled [1-13C]glycine (1a) in 52% overall yield and from [2-13C]glycine (1b) in 56% overall yield, respectively. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[N(C2H3)3]choline (9) was prepared from 2-aminoethanol in 39% overall yield. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[alpha-C2H2]choline (12) was prepared from N,N-dimethylglycine ethyl ester in 50% overall yield.

Structural and thermotropic properties of synthetic C16:0 (palmitoyl) ceramide: effect of hydration.

Differential scanning calorimetry (DSC) and X-ray diffraction techniques have been used to investigate the structure and thermotropic properties of synthetic, non-hydroxy fatty acid (16:0) ceramide (NFA(C16)CER) as a function of hydration. Anhydrous NFA(C16)CER shows a single, broad endothermic transition at 95.4 degrees C (delta H = 10.4 kcal/mol). On hydration, a broad exothermic transition appears at approximately 50-70 degrees C while the main endothermic transition decreases to 90.0 degrees C (delta H = 13.8 kcal/mol). The enthalpy of the exothermic transition increases with hydration to a maximum value, delta H = 4.8 kcal/mol. This polymorphic phase behavior depends on the low temperature incubation time and prior cooling rate. X-ray diffraction of fully hydrated NFA(C16)CER at 26 degrees C, shows a well-ordered lamellar phase with a bilayer periodicity d = 46.9 A. At 68 degrees C, above the first exothermic transition, X-ray diffraction shows again a lamellar phase with reduced bilayer periodicity d = 41.8 A and an increased number of both lamellar and wide-angle reflections indicative of enhanced layer and chain packing order, respectively. At 90.0 degrees C, above the main transition, the diffraction pattern shows a broad, intense reflection at 29.9 A and a diffuse reflection at 4.6 A, indicative of a melted chain phase. On cooling, NFA(C16)CER exhibits polymorphic phase behavior involving the conversion of the melted chain phase to a metastable bilayer phase. On heating, this metastable phase undergoes an exothermic transition to a stable bilayer phase; on further heating, NFA(C16)CER converts endothermically to the melted-chain phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Single crystal X-ray structures of the two 4-heptadecyl derivatives of (1R,5S)-3,6,8-trioxabicyclo[3.2.1]octane.

The single crystal structures of the two diastereomeric 4-heptadecyl derivatives of (1R,5S)-3,6,8-trioxabicyclo[3.2.1]octane have been determined by X-ray diffraction to be (1R,4R,5S)-heptadecyl-3,6,8-trioxabicyclo[3.2.1]octane (I) and (1R,4S,5S)-4-heptadecyl[3,6,8-trioxabicyclo[3.2.1]octane (II), respectively, which have an exo or axial 4-heptadecyl group, and an endo or equatorial 4-heptadecyl group, respectively. The structures of I and II had been suggested by their phase-sensitive 2D NOESY 1H-NMR spectra, but are now established unambiguously. These optically pure non-ionic lipid-like amphipathic molecules (I and II) represent the first 3,6,8-trioxabicyclo[3.2.1]octanes for which single crystal structures have been solved. Crystals of both isomer I and isomer II were orthorhombic with space group P2(1)2(1)2(1), and had unit cell dimensions of a = 9.586, b = 43.14, c = 5.289 A, and a = 7.34, b = 51.8, c = 5.636 A, respectively. The structures of I and II were both solved by using direct methods to R = 0.045 and R = 0.086, respectively. Both I and II pack in stacked bilayers with interdigitating and tilting hydrocarbon chains. The molecular and hydrocarbon cross sections are I: S = 50.70 A2, sigma = 19.00 A2; and II: S = 41.37 A2, sigma = 18.26 A2.

Syntheses of racemic and nearly optically pure ether lipids and evaluation of in vitro antineoplastic activities.

In addition to rac-2-O-methyl-1-O-octadecylglycero-3-phosphocholine (rac-ET-18-OCH3, rac-Edelfosine, 1), three racemic ether lipid analogs, 4, 5, and 6, were synthesized where N,N-dimethylamino, N-methylpyrrolidino, and N-methylmorpholino groups, respectively, have been substituted for the trimethylammonio group. The two enantiomers, (R)-ET-18-OCH3 (2) and (S)-ET-18-OCH3 (3), and all four possible chiral methylcholine analogs, 7, 8, 9, and 10, of (R)-ET-18-OCH3 (2) were also synthesized. Three human leukemic cell lines (CEM, HUT 78, and Namalwa) were used to assess the in vitro antineoplastic properties of these 10 ether lipid analogs. At ether lipid concentrations of 5-50 micrograms/mL, dose- and time-dependent cytotoxicities were demonstrated up to 24 h. CEM and HUT 78, both T cell derived, were more sensitive to the ether lipids than Namalwa, which is B cell derived. rac-ET-18-OCH3 (1) with its R and S enantiomeric forms, 2 and 3, respectively, exhibited modest stereoselectivity in HUT 78 and Namalwa with 1 and 2 slightly more cytotoxic than 3. Ether lipid (EL) analogs 4, 5, and 6 demonstrated significantly greater cytotoxicity in normal peripheral lymphocytes, 4 and 6 exhibited a modest increase in cytotoxicity in HUT 78 and Namalwa (P < 0.05), and 5 demonstrated greater cytotoxicity (P < 0.05) in Namalwa than the parent EL 1. The calculated 24 h ID50 values suggest that the beta-methyl analogs, 9 and 10, were more cytotoxic than the alpha-methyl analogs, 7 and 8, in all the tested cancer cell lines.

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).

Synthesis of 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine (PHPC).

A general method for the chirospecific synthesis of 1-acyl-2-alkyl-sn-glycero-3-phosphocholines is described. 1-Palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine (PHPC) was synthesized in 18% overall yield in ten steps via five new synthetic intermediates, and 1-acetyl-2-hexadecyl-sn-glycero-3- phosphocholine (AHPC) was also synthesized. 1-Acyl-2-alkyl-sn-glycero-3-phosphocholines, which have not been found to exist in nature, are ether lipid analogs of 1,2-diacyl-sn-glycero-3-phosphocholines, which are important components of cell membranes. Biophysical studies of hydrated bilayers of PHPC will be of interest in probing the critical importance of the central region of these amphiphilic molecules to the molecular assemblies that are formed.

Debenzylation and detritylation by bromodimethylborane: synthesis of mono-acid or mixed-acid 1,2- or 2,3-diacyl-sn-glycerols.

A novel method of deprotecting primary alcohols protected with either benzyl or trityl groups by using bromodimethylborane under mild reaction conditions (dichloromethane, -20 to 5 degrees C) has been applied to the synthesis of optically pure mono-acid or mixed-acid 1,2- or 2,3-diacyl-sn-glycerols. This method was particularly useful for the synthesis of long saturated acyl (C12 to C24) as well as unsaturated diacyl-sn-glycerols since little or no acyl migration occurred during deprotection. Diacylation of 3-benzyl-sn-glycerol or 1-benzyl-sn-glycerol followed by bromodimethylborane debenzylation gave mono-acid 1,2- or 2,3-diacyl-sn-glycerols, respectively. The mixed-acid 1,2- or 2,3-diacyl-sn-glycerols were prepared from 1-acyl-sn-glycerols or 3-acyl-sn-glycerols, respectively, by tritylation, acylation with a different fatty acid, followed by detritylation with bromodimethylborane.

Comparison of oral bioavailability of two dosage-forms of progesterone in women.

For poorly water soluble drugs, the dissolution process in biological fluids the rate limiting step in absorption. However, the utilization of some galenic processes such as solid dispersions (SD) leads to an improvement in quality and intensity of the drug gastro-intestinal absorption. In a previous work, the in vitro studies of the dissolution curves of both the pure micronized progesterone (MP) and the progesterone-PEG 6000 SD revealed marked increases in the progesterone dissolution rates for all the SD investigated compared to the pure MP. The aim of this work was to investigate the in vitro results after oral administration of the two pharmaceutical forms to menopaused volunteer women.

Determination of alpha-bisabolol and d-panthenol in cosmetic products by gas chromatography.

Synopsis alpha-bisaboloi and d-panthenol are used in many cosmetic preparations, respectively, for their anti-inflammatory and regenerating properties. Their quantitative determination was an important element of their evaluation in these emulsions. Their concentration has been determined by gas chromatographic techniques, using a flame ionization detector. In both cases, the internal standards have been chosen for their compatibility with the analysis of extracted substances. Owing to the complexity of the cosmetic formulations, a preliminary extraction of alpha-bisabolol and d-panthenol was necessary. For the two substances the preparative separation was based on a liquid-liquid extraction. After dissolving the emulsion in methanol and diluting it with an aqueous buffer solution alpha-bisabolol was then extracted by ethyl acetate and d-panthenol by ethyl formate.

Alterations in red blood cell sugar transport by nanomolar concentrations of alkyl lysophospholipid.

Acyl lysolipids presented in vitro to red blood cells in amounts comparable to blood serum levels inhibit protein-mediated glucose transport (Naderi, A., Carruthers, A. and Melchior, D.L. (1989) Biochim. Biophys. Acta 985, 173-181). In this study, an alkyl lysolipid (2-O-methyl-1-O-octadecyl-sn-glycero-3- phosphocholine; ALP), was found to be an order of magnitude more effective in inhibiting sugar transport than the most potent acyl lysolipid. Bilayer concentrations of ALP as low as 5 ALP molecules per transporter (0.1 mol% of total membrane lipid) result in a 50% inhibition of transport activity. ALP acts as a competitive inhibitor of exchange L-glucose transport, of CCB binding to the glucose transporter and of D-glucose inhibition of CCB binding to the transporter. Inhibition of zero-trans sugar uptake by ALP is noncompetitive. The two enantiomers of ALP show a different ability to inhibit sugar transport. The action of ALP is consistent with a mechanism in which ALP interacts with a transmembrane portion of the sugar transport molecule resulting in a competitive displacement of D-glucose or cytochalasin B from the cytosolic facing side of the transport molecule. The simplest explanation of our findings is a direct interaction of the ALP molecule with the transport protein.