RIDME distance measurements using Gd(iii) tags with a narrow central transition.

Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.

Compact, hydrophilic, lanthanide-binding tags for paramagnetic NMR spectroscopy.

The design, synthesis and evaluation of four novel lanthanide-binding tags for paramagnetic NMR spectroscopy are reported. Each tag is based on the ((2S,2'S,2''S,2'''S)-1,1',1'',1'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(propan-2-ol)) scaffold, featuring small chiral alcohol coordinating pendants to minimise the size and hydrophobic character of each tag. The tags feature different linkers of variable length for conjugation to protein via a single cysteine residue. Each tag's ability to induce pseudocontact shifts (PCS) was assessed on a ubiquitin A28C mutant. Two enantiomeric tags of particular note, C7 and C8, produced significantly larger Δχ-tensors compared to a previously developed tag, C1, attributed to the extremely short linker utilised, limiting the mobility of the bound lanthanide ion. The C7 and C8 tags' capacity to induce PCSs was further demonstrated on GB1 Q32C and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) S112C/C80A mutants. Whilst factors such as the choice of lanthanide ion, pH and site of conjugation influence the size of the PCSs obtained, the tags represent a significant advance in the field.

Direct measurement of electrical conductance through a self-assembled molecular layer.

The self-assembly of organic molecules on surfaces is a promising approach for the development of nanoelectronic devices. Although a variety of strategies have been used to establish stable links between molecules, little is known about the electrical conductance of these links. Extended electronic states, a prerequisite for good conductance, have been observed for molecules adsorbed on metal surfaces. However, direct conductance measurements through a single layer of molecules are only possible if the molecules are adsorbed on a poorly conducting substrate. Here we use a nanoscale four-point probe to measure the conductivity of a self-assembled layer of cobalt phthalocyanine on a silver-terminated silicon surface as a function of thickness. For low thicknesses, the cobalt phthalocyanine molecules lie flat on the substrate, and their main effect is to reduce the conductivity of the substrate. At higher thicknesses, the cobalt phthalocyanine molecules stand up to form stacks and begin to conduct. These results connect the electronic structure and orientation of molecular monolayer and few-layer systems to their transport properties, and should aid in the rational design of future devices.

Hydrogen-bonded PTCDA-melamine networks and mixed phases.

A stable hydrogen-bonding junction is formed between 3,4,9,10-perylene-3,4,9,10-tetracarboxylic-dianhydride (PTCDA) and 1,3,5-triazine-2,4,6-triamine (melamine). This bimolecular system was studied on the Ag-Si(111) square root 3 x square root R 30 degrees surface at sub-monolayer coverage, and two distinct phases are observed. A hexagonal lattice is formed that is stabilized by hydrogen bonding between PTCDA and melamine. This phase, in which melamine acts as a 3-fold vertex, is a close analogue to the 3,4,9,10-perylene-3,4,9,10-tetracarboxylic-diimide-melamine network reported recently. To our knowledge this hydrogen-bonding junction has not been previously observed and might not be expected due to lone pair repulsion. However we confirm that this combination is stable using ab initio methods. In the second intermixed phase parallel rows of PTCDA molecules coexist with an array of melamine molecules, and we propose a model for this structure.

Square, hexagonal, and row phases of PTCDA and PTCDI on Ag-Si(111)square root(3) x square root(3)R30 degrees.

We have investigated the ordered phases of the perylene derivatives perylene-3,4,9,10-tetracarboxylic-3,4,9,10-dianhydride (PTCDA) and the imide analogue PTCDI on the Ag-Si(111)square root(3) x square root(3)R30 degrees surface using scanning tunneling microscopy. We find that PTCDA forms square, hexagonal, and herringbone phases, which coexist on the surface. The existence of a square phase on a hexagonal surface is of particular interest and is a result of a near commensurability between the molecular dimensions and the surface lattice. Contrast variations across the square islands arise from PTCDA molecules binding to different sites on the surface. PTCDI on Ag-Si(111)square root(3) x square root(3)R30 degrees forms extended rows, as well as two-dimensional islands, both of which are stabilized by hydrogen bonding mediated by the presence of imide groups. We present models for the molecular arrangements in all these phases and highlight the role of hydrogen bonding in controlling this order.

Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius.

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

The three-dimensional structure of the Nudix enzyme diadenosine tetraphosphate hydrolase from Lupinus angustifolius L.

The solution structure of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap4A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet and three helices (alphaI, alphaIII, alphaIV). The root-mean-squared deviation for the backbone (C',O,N,Calpha) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower definition, particularly an isolated helix (alphaII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between alphaII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded beta-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

Assessment of value and applications of in vitro testing of topical dermatological drug products.

The FDA recently issued a guidance covering practices of scaleup and post approval changes with semisolids (SUPAC-SS). This guidance outlines the steps that must be taken by a company to maintain certification of its semisolid dermatological products after quantitative changes have been made in their compositions and/or after changes have been made in the sourcing of their key ingredients, in their processing, in their batch sizes, and/or after their site of manufacture has been relocated. A key element within the guidance is a release test to be used to determine if the diffusional release of a drug found in a formulation is the same after changes have been made to the formulation as it was prior to implementing the changes. The AAPS-FDA sponsored workshop was set up to explore this qualifying test. The stated aims of the workshop were: a) to illustrate the methodology and techniques of in vitro release testing, b) to show the sensitivity of in vitro release with respect to manufacturing variables and to variations in components and composition (of specific formulations), c) to recognize in vitro release testing as a useful procedure for SUPAC documentation, d) to highlight and evaluate other applications of in vitro release testing, e) to explore the degree to which in vitro release testing and bioavailability may be related, and f) to evaluate the role of in vitro release testing of topical dosage forms as a tool to improve product quality.

Absorption of sulindac from a novel (Pro-SorbTM) liquid formulation.

The absorption of sulindac from two different 200 mg oral dosage forms, Pro-SorbTM liquid and Clinoril tablets, was compared following administration to eight healthy human volunteers. Subjects received both formulations according to a randomized crossover design and blood samples were collected at selected times during 24 h. Concentrations of both sulindac and its active sulphide metabolite were determined by HPLC and individual serum concentration versus time profiles were constructed. Maximal serum concentrations (Cmax) and area under the curve values (AUClast and AUC0-infinity) were compared for both sulindac and sulindac sulphide. The results indicate that the rate and extent of absorption of sulindac from Pro-SorbTM liquid are significantly greater than those from Clinoril tablets, a finding consistent with the observation that the liquid form produces less gastric irritation following oral administration.

Drug transport across nylon 610 films: influence of synthesis variables.

Nylon 610 is a hydrophilic polymer with considerable potential as a membrane for drug microencapsulation. To better understand drug transport through such membrane, the influence of the solvents and monomers used in the synthesis of nylon films were examined using a full factorial study. Nylon 610 films were synthesized by an interfacial polycondensation reaction using hexamethylenediamine (HD) in the water phase and sebacoyl chloride (SC) in the organic phase, which was a solvent blend of chloroform and trichlorotrifluoroethane at ratios of 1:1, 1:4, and 4:1. Monomer concentrations studied were 0.2, 0.4, and 0.6 M with respect to their appropriate phase, while the monomer ratios were 1:1, 3:1, and 1:3. The molecular weight, porosity, thickness, and crystallinity of the films were characterized. The transport of potassium chloride, hydrocortisone, and m-cresol was studied at 25 degrees C as a function of the synthesis variables. Potassium chloride was selected to measure the porosity of the membrane. Hydrocortisone and m-cresol, a known solvent for nylon 610, were used to study pore and solution-diffusion transport, respectively. The molecular weight of the films was proportional to the chloroform concentration. As the molecular weight increased, film thickness, porosity, and hydrocortisone permeability increased. As the molecular weight decreased, film thickness and porosity decreased, while m-cresol permeability increased. These results can be explained on the basis of HD ability to readily partition into a good solvent such as chloroform permitting high molecular weight polymer to form before precipitation.

Albumin microspheres as a drug delivery system: relation among turbidity ratio, degree of cross-linking, and drug release.

The degree of cross-linking of albumin microspheres, with and without drug, was assessed using turbidity measurements carried out in the presence of water and the protein denaturant guanidine hydrochloride (GuHCl) at a concentration that disrupted noncovalent bonds while having no effect on covalent bonds. The measurements allowed calculation of a turbidity ratio (TG/TW), expressed as the ratio of the turbidity of albumin microspheres in 6 M GuHCl (TG) divided by that in water (TW). A linear relation existed between TG/TW and the (i) temperature at which the microspheres were prepared, (ii) concentration of the cross-linking agent glutaraldehyde, and (iii) time of exposure to a second cross-linking agent, formaldehyde vapor, three conditions that increase the degree of cross-linking. The turbidity ratio also increased as the concentration of the albumin solution used to prepare the microspheres increased from 25 to 50%. Drug release from the microspheres consisted of an initial, rapid, burst followed by a second, slower, phase. The rates in both release phases were inversely related to the turbidity ratio, suggesting that this parameter has utility as an indicator of the degree of cross-linking in albumin microspheres.

The influence of liquid crystalline phases on drug percutaneous absorption. I. Development of a vehicle.

A phase diagram approach has been used to formulate topically applied vehicles containing liquid crystalline phases. The current paper describes the construction of a major portion of the polyoxyethylene(20)cetyl ether:dodecanol:water phase diagram. Known mixtures of the three components were equilibrated and centrifuged to separate the resultant conjugate phases. These were identified and analyzed quantitatively to determine phase boundaries in relevant portions of the phase diagram. Two isotropic liquid phases, several two- and three-phase regions, a solid surfactant phase, and at least three distinct liquid crystalline phases were identified. The determination of tie lines was undertaken in a two-phase region containing an aqueous isotropic micellar solution and a liquid crystalline gel. This information will be used to prepare a number of vehicles of known phase composition and concentration for a systematic evaluation of the effect of liquid crystalline phases on transdermal drug delivery.

The influence of liquid crystalline phases on drug percutaneous absorption. II. Permeation studies through excised human skin.

The influence of liquid crystalline (LC) phases on the percutaneous absorption of a model compound (ploxicromil; PXC) was studied with the use of the phase diagram for the surfactant, oil, and water comprising the vehicles. Two separate sets of vehicles, representing two different tie lines lying in the L1 + LC phase region, were prepared in which the concentration of LC was varied over the range 0 to 100% along each tie line. In vitro permeation studies of PXC from these systems were conducted using excised human skin and the flux values determined as a function of the percentage LC present in the vehicles. In virtually all cases, the flux reached a peak at 5-10% LC and then decreased significantly as the fraction of LC present increased further. The pattern of behavior observed is discussed in terms of current theories describing membrane-controlled and vehicle-controlled diffusion, none of which adequately model the results obtained.

Effects of ionization on the percutaneous absorption of drugs: partitioning of nicotine into organic liquids and hydrated stratum corneum.

To gain further insight into the disposition of ionizable drugs in the stratum corneum, the partitioning of nicotine as a function of pH was studied in excised, hydrated, human stratum corneum (SC) and the organic liquids n-butanol, n-octanol, isopropyl myristate, and Miglyol 812. Partitioning in n-octanol, isopropyl myristate, and Miglyol 812 was consistent with the pH-partition hypothesis, while partitioning in n-butanol agreed with agreed with a model for the partitioning of the free base and an ion pair which dissociates in the organic phase. The results in SC also suggested the partitioning of ion pairs. Binding studies indicated that neither the un-ionized nor the ionized species is bound significantly in the stratum corneum. Trichloroacetate (TCA) anion increased partitioning of ionized nicotine in n-butanol, but had no effect in stratum corneum. Delipidization of the stratum corneum decreased the partition coefficient for the free base, but had no effect on the ionized species. Thermodynamic parameters determined from van't Hoff plots were consistent with the entry of un-ionized nicotine into ordered lipids. These results suggest that the un-ionized form is found within the lipid regions of the stratum corneum, while the ionized form is located in aqueous regions.

Sustained-release delivery systems, II: In vitro dissolution of 4,4'-sulfonylbisbenzamine (dapsone)--N-dodecanoyl-4,4'-sulfonylbisbenzamine comelts.

An approach under investigation in our laboratory is the development of slowly dissolving particles containing an active drug, mixed or coated with a less soluble derivative or derivatives, that may hydrolyze back to the active drug following solution. Such particles have potential for sustained release by a number of routes of administration, including intramuscular injection. In the present work, the in vitro dissolution of particles of 4,4'-sulfonylbisbenzamine (dapsone) (1) and various comelts of 4,4'-sulfonylbisbenzamine (dapsone) (1) and N-dodecanoyl-4,4'-sulfonylbisbenzamine (the monolauryl derivative of dapsone) (3) was studied using a continuous flow-through cell under laminar flow conditions. Dissolution of particles of dapsone showed a biphasic pattern when plotted according to the Hixson-Crowell cube root equation. Dissolution rates of dapsone (1) from the comelts were found to correlate with the physical state of 1 in the solid binary dispersion. Comelts of 1:3 with a content higher than 36.5% w/w 1 (the eutectic mixture) had initial rates of dissolution which appeared to be dependent on the porosity of the particles. However, once this initial phase passed, the total amount dissolved increased as the amount of noneutectic 1-rich solid solution in the comelt increased. This suggests that 1 in the eutectic mixture has a slower dissolution rate than when present in the noneutectic, or excess 1-rich solid solution form. The comelts with a content of dapsone at or below the eutectic mixture dissolved more slowly and their dissolution rates were less dependent on concentration of 1. The mechanism of release of 1 from 1:3 comelts appeared to be matrix diffusion-controlled.

Sustained-release delivery systems, I: Phase diagram studies of dapsone and selected derivatives.

In order to develop slowly dissolving particles containing the antileprotic drug dapsone (4,4'-sulfonylbisbenzamine, that would be suitable for a sustained-release intramuscular injection, the dilauryl and monolauryl derivatives of dapsone, N,N'-didodecanoyl-4,4'-sulfonylbisbenzamine and N-dodecanoyl-4,4'-sulfonylbisbenzamine, respectively, were studied for their ability to form solid dispersions with the parent compound. The 1:2 binary phase diagram showed these two compounds were partially miscible in the liquid state, leading to the coexistence of a monotectic and a eutectic system in the phase diagram. The 1:3 phase diagram showed that these compounds were completely miscible in the liquid state and formed discontinuous solid solutions in the solid state. At a cooling rate of 2.5 degrees C min-1, eutectic mixtures lying on the dapsone side of the eutectic point formed glass solutions. Taken as a whole, the results demonstrate that the molecular interactions between 1 and 3 are stronger than those between 1 and 2. Accordingly, 3 would appear to be the better carrier for reducing the rate of dissolution of dapsone from a solid dispersion.

Influence of ophthalmic formulations on sodium cromoglycate disposition in the albino rabbit eye.

The effect of different ophthalmic vehicles on the disposition of sodium cromoglycate in tears and ocular tissues of the rabbit eye has been studied over 6 h. The vehicles contained sodium cromoglycate, 2% in an aqueous solution, 2 and 4% in an oleaginous formulation of polyethylene and mineral oil (Plastibase 5W), and 4% in an absorption ointment base of 10% hypoallergenic acetylated lanolin (Modulan) in paraffins. The last formulation was superior to all others studied over 6 h in prolonging the retention of sodium cromoglycate in the precorneal area and the conjunctiva. The concentration of sodium cromoglycate in the tears, conjunctiva and cornea 6 h after administration of the acetylated lanolin base equalled or exceeded the concentrations obtained with the aqueous solution 1 h post-instillation.

Drug permeation through human skin II: Permeability of ionizable compounds.

The aim of this study was to establish whether ionized as well as un-ionized forms of certain 4-oxo-4H-1-benzopyran-2-carboxylic acids (chromone-2-carboxylic acids) with pKa values less than 2 permeated through excised human skin and, if so, to determine the permeability coefficients of the permeating species. The permeation properties of four carboxylic acids were studied as a function of concentration over the pH range 5-7 at 37 degrees C with plexiglass diffusion cells. Plots of J/CA- (the total flux due to un-ionized and ionized species obtained under steady-state conditions per unit concentration of ionized drug in the donor compartment) against CH3O+ resulted in straight-line relationships. The intercepts of these plots were shown to equal PA-, whereas the slopes multiplied by the Ka values of the compounds equalled PHA, the permeability coefficients of the ionized and un-ionized species, respectively. With all four compounds, both species were found to permeate skin, although the permeability coefficients of the un-ionized species were approximately 10(4) greater than those for the ionized species. It was demonstrated that the relative contributions of the ionized and un-ionized species to the total flux, as well as the total flux, vary significantly, depending on the pH of the drug solution in the donor cell. This may provide a means of controlling the flux of these and similar compounds through human skin.