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A hydrate of co-crystal of piracetam and 3,5-dihydroxybenzoic acid was obtained via crystallization from water. Single-crystal X-ray data show that piracetam/3,5-dihydroxybenzoic acid tetrahydrate (P35TH) crystallizes in the triclinic system with a P1 space group. The physicochemical properties of co-crystal hydrate were characterized using powder X-ray diffractometry, differential scanning calorimetry (DSC), thermogravimetric analyzer (TGA), and FTIR spectroscopy. The dehydration kinetics of P35TH was monitored at various temperatures and heating rates by DSC and TGA. Activation energy of P35TH dehydration was obtained using temperature ramp DSC, isothermal and nonisothermal TGA methods. Kinetic analysis of isothermal TGA data was fitted to various solid-state reaction models. Mechanistic models derived from isothermal dehydration kinetic data are best described as a 2-dimensional diffusion mechanism. A correlation was noted between the dehydration behavior and the bonding environment of the water molecules in the crystal structure. This study is a good demonstration of complexity of co-crystal hydrate and their dehydration behavior.

A graphite oxide (GO)/heparin–benzalkonium chloride (C12) composite was synthesized. The composite was characterized by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). XRD data showed that spacing between layers of GO increased from 0.59 to 3.256 nm. This enlarged layer spacing suggested that heparin–C12 complex intercalated completely in between layers of GO. FTIR also confirmed intercalation of heparin–C12 complex into GO gallery. In vitro release rate of heparin from GO–heparin–C12 was monitored for 30 days. Heparin released at a very fast rate from the composite matrix in the first day. The release slowed down significantly after the first day and continued for 30 days. In addition, antibacterial activity of the composite against Escherichia coli (E. coli) and Staphlococcus aureus (S. aureus) was evaluated using zone of inhibition and colony count assays. Both GO–heparin–C12 and GO–C12 clearly showed antibacterial activity against E. coli and S. aureus while GO alone has a relatively low activity against S. aureus and almost no effect on E. coli.

The aim of this study was to investigate the effect of the concentration of the active pharmaceutical ingredient on the physical state of mannitol in frozen aqueous systems.A human monoclonal antibody was used as the model protein. Mannitol and sucrose were used as the bulking agent and the lyoprotectant, respectively. The thermal behavior of frozen mannitol-sucrose solutions during and after annealing, in the absence and presence of the protein, were characterized by low-temperature powder X-ray diffractometry and differential scanning calorimetry. The influence of the protein on the crystallization behavior of mannitol was also evaluated.The excipient concentration had a pronounced effect on the glass transition temperature of maximally freeze-concentrated amorphous phase (T(g)'). At fixed excipient compositions, the protein had no effect on the T(g)' if the protein concentration wasor =20 mg/ml. However, at higher protein concentrations, there was a marked increase in T(g)' as a function of protein concentration. The inhibitory effect of the protein on mannitol crystallization was concentration dependent and was directly evident from X-ray diffractometry experiments. Annealing facilitated both mannitol nucleation and crystal growth even in the presence of the protein.The ratio of mannitol to sucrose and the protein concentration have an impact on the T(g)' and may therefore influence the primary drying temperature. The protein inhibits both the nucleation and growth of mannitol crystals and this effect seems to be concentration dependent. The presence of the protein and the protein concentration dictate the processing conditions, i.e., annealing time, annealing temperature, and primary drying temperature.

The purpose of this work was to evaluate nanoindentation as a means to characterize the material properties of pharmaceutical solids. X-ray diffraction of potassium chloride and acetaminophen showed that samples prepared by cooling a melt to a crystalline sample as opposed to slow recrystallization had the same crystal structure. With analysis of the force-displacement curves, the KCl quenched samples had a hardness that was 10 times higher than the recrystallized KCl, while acetaminophen quenched samples were 25% harder than the recrystallized samples. The elastic moduli of the quenched samples were also much greater than that observed for the recrystallized samples. Although the elasticity was independent of load, the hardness increased with load for acetaminophen. With each sample, the flow at constant load increased with applied load. Etching patterns obtained by atomic force microscopy showed that the KCl quenched sample had a higher dislocation density than the recrystallized sample, although there was no evident difference in the acetaminophen samples. Overall, the differences in the observed sample properties may be related to the dislocation density. Thus, nanoindentation has been shown to be a sensitive method for determining a processed-induced change in the hardness, creep, and elasticity of KCl and acetaminophen.

The force-displacement profiles of four well-characterized materials that represent both soft/hard and plastic/brittle materials have been obtained using a novel nanoindentation technique. Flat surfaces of acetaminophen, potassium chloride, sucrose, and sodium stearate were prepared by melting or recrystallization, and the melting points were measured. Topographic and the corresponding first derivative images were obtained both before and after indentation. The materials were indented using a 10 s loading time, followed by a 2 s hold, and ending with a 10 s unloading time thereby providing a unique force-displacement profile for each material. The loading profile of acetaminophen was discontinuous, whereas the profiles for the other three materials were smooth. The profiles were analyzed and the rank order of hardness was sucrose > acetaminophen > KCl > sodium stearate, which is consistent with the literature. The rank order of the stiffness, which is related to the modulus of elasticity, was sucrose > KCl > acetaminophen > sodium stearate. Given the flexibility and power of this approach, nanoindentation coupled with atomic force microscopy may be a useful means to characterize materials for evaluating tablet-processing conditions, perhaps at a preformulation stage.

The inhalation delivery of difluoromethylornithine (DFMO) was evaluated to determine its feasibility for use in lung cancer chemoprevention. Aerosol droplets of DFMO were produced, and the solvent was removed by a reflux drying column. Equivalent doses of DFMO (40 mg/kg) were given over 10 minutes by inhalation and by gavage, and the serum and tissue levels were determined. Inhalation of DFMO resulted in a peak lung concentration 80 times higher and area under the curve (AUC) that was 35 times higher than oral dosing. The estimated pharmacokinetic advantage based on serum and lung AUCs was 13 in favor of inhalation.

The association of hydrophobic, cationic drugs with lung surfactant was determined to assess the pharmacokinetic implications on drug disposition and retention in the lung.The distribution coefficients, K, were determined at 25 and 37 degrees in normal saline solution buffered at pH 7.4 for a series of structurally related, cationic drugs. Drugs were dispersed into lung surfactant, equilibrated, and then centrifuged to separate the aqueous phase from the surfactant pellet. Drug concentrations in the supernatant and pellet were determined following dilution using spectrophotometric assays. In addition, the apparent acid dissociation constant of quinacrine in the presence and absence of surfactant was determined by measuring the pH-dependent absorption spectra. The effect of stereochemistry on the distribution of drugs into surfactant was examined with (R)- and (S)-propranolol.The mole fraction distribution coefficients for amitriptyline, promethazine, promazine, ethopropazine, imipramine, R-propranolol, and S-propranolol at 25 degrees C were 6,560 +/- 500, 28,400 +/- 1,500, 12,100 +/- 840, 5,480 +/- 330, 4,490 +/- 250, 8,680 +/- 260, 8,190 +/- 530, respectively. At 37 degrees C, the distribution coefficients were generally smaller indicating a significant exothermic heat of transfer for these solutes from aqueous solution to the lung surfactant. The pKa of quinacrine was 7.43 +/- 0.04 in aqueous solution and was shifted to 7.62 +/- 0.06 in the presence of lung surfactant. From this shift, the double layer potential for quinacrine-lung surfactant was estimated to be -0.012 V assuming a dielectric constant equivalent to that of water.Cationic drugs have very favorable distributions from an aqueous solution to the lipid phase of lung surfactant. The transfer process generally has both a large entropic and enthalpic contribution. The latter thermodynamic aspect may be related to the charge interaction between the solute and the negatively charged surfactant. Finally, no significant effect of stereochemistry was evident with the distribution of (R)- and (S)-propranolol.

High-resolution tapping-mode atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) are used to study nonstoichiometric polyelectrolyte−surfactant complexes (PSCs) formed between poly(vinyl sulfate) and alkyltrimethylammonium bromide (CnTAB). The morphological evolution of PSC films prepared with different surfactant loadings (i.e., different surfactant/anionic-site stoichiometries) is explored as a function of surfactant chain length (n = 12, 14, 16, 18). PSC films were prepared with 1%, 10%, 25%, and 50% surfactant loading. At the lowest loading, relatively uniform films are obtained, despite the fact that the surfactant concentrations in the solutions employed were all above the critical aggregation concentration. At 10% loading, for the longer chain surfactants (n = 16 and 18), small protrusions (10−40 nm in height) appear in the films. These are attributed to the formation of spheroidal and/or discoidal polyelectrolyte−surfactant micelles. For films of 25−50% surfactant loadi...

Thin films prepared from a complex formed between poly(vinyl sulfate) (PVS) and a cationic indocarbocyanine surfactant dye (DiI) are investigated by near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM). Local film morphology and chemical composition are studied as a function of dye/anionic-site stoichiometry. AFM images show that overall film morphology changes modestly with dye content. Fluorescence NSOM images reveal that at lowest PVS loading, the films are extremely heterogeneous, exhibiting round fluorescent regions 100−250 nm in size. As the dye content is increased, the fluorescent regions increase in number but remain of similar sizes. NSOM fluorescence spectra prove these regions contain aggregated DiI and indicate they are likely polymer−surfactant micelles entrapped in the films.

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.

The local optical and electrooptical properties of dye-doped polymer-dispersed liquid crystal (PDLC) thin films are explored by near-field scanning optical microscopy (NSOM). Detailed information on the mechanisms for dye alignment and electric-field-induced dye reorientation in localized sample regions is sought. Dye reorientation is predicted to occur by two distinct processes: (i) via its interactions with the liquid crystal host and (ii) via the direct interactions of its permanent and induced dipole moments with the applied field. The liquid crystal is doped with a nearly isotropically aligned BODIPY dye so that contributions of both reorientation mechanisms can be explored. An order parameter of 0.10 ± 0.02 is measured for BODIPY in bulk aligned samples, suggesting that host−guest interactions are very weak. Simultaneously recorded birefringence (transmission) and fluorescence NSOM images confirm that the dye is apparently not well aligned, even on submicrometer length scales. Surprisingly, NSOM st...

We describe a strategy for the incorporation of a 2'-C-branched ribonucleoside, 2'-C-beta-methylcytidine, into oligonucleotides via solid-phase synthesis using phosphoramidite derivatives. 4-N-Benzoyl-2'-C-beta-methylcytidine (2b) was synthesized by coupling persilylated 4-N-benzoylcytosine with 1,2,3,5-tetra-O-benzoyl-2-C-beta-methyl-alpha-(and beta)-D-ribofuranose (1) in the presence of SnCl(4) in acetonitrile, followed by selective deprotection with NaOH in pyridine/methanol. The 3'- and 5'-hydroxyl groups were blocked as a cyclic di-tert-butylsilanediyl ether 3 by treatment with di-tert-butyldichlorosilane/AgNO(3) in DMF. The 2'-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by treatment with tert-butylmagnesium chloride followed by addition of tert-butyldimethylsilyl trifluoromethanesulfonate in THF. As an alternative to 2'-silyl protection, the corresponding 2'-O-tetrahydropyranyl ether 4b was prepared by treatment of 3 with 4,5-dihydro-2H-pyran in the presence of a catalytic amount of 10-camphorsulfonic acid in methylene chloride. The di-tert-butylsilanediyl groups of 4a and 4b were removed by treatment with pyridinium poly(hydrogen fluoride) to afford 5a and 5b, respectively. Protection of the 5'-hydroxyl group as a dimethoxytrityl ether and phosphitylation of the 3'-hydroxyl group by the standard procedure gave the phosphoramidite derivatives 7a and 7b. Both 7a and 7b could be used to incorporate 2'-C-beta-methylcytidine into oligonucleotides efficiently via standard solid-phase synthesis, but the tetrahydropyranyl group of 7b was more readily removed from oligonucleotides than the tert-butyldimethylsilyl group of 7a. Oligonucleotides containing 2'-C-beta-methylcytidine undergo base-catalyzed degradation analogous to natural RNA.

The effect of position isomerism on the co-crystals formation and physicochemical properties was evaluated. Piracetam was used as the model compound. Six position isomers, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic acid (DHBA), were used as the co-crystal formers. Co-crystals were prepared on a 1:1 molar ratio by crystallization from acetonitrile. The solid-state properties of co-crystals were characterized using X-ray powder diffractometry (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR). All co-crystal formers formed co-crystal with piracetam except 2,6-DHBA. This failure was possibly due to steric hindrance of two bulk hydroxyl groups and preference of intra-molecular hydrogen bonding formation between hydroxyl group and carboxylic acid group. The XRD patterns of resulting co-crystal indicated that they are highly crystalline and different than parental compounds. Based on the single crystal data, P_23DHBA is orthorhombic while P_24DHBA, P_34DHBA, and P_35DHB belong to monoclinlic system. The hydrogen bonding network patterns of the co-crystals are also different. DSC data showed that the melting temperatures of resulting co-crystals are all lower than that of the starting materials. The melting point rank order of the co-crystals is: P_24DHBA > P_34DHBA > P_23DHBA > P_25DHBA > P_35DHBA. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:246–254, 2010

The aim of this study was to relate the tableting performance assessed by an instrumented tableting machine to the mechanical properties measured by nanoindentation. Three different polymorphic forms of sulfathiazole were prepared by recrystallization, and the density and X-ray powder diffraction patterns were measured and compared with theoretical density and simulated powder patterns, respectively. Tablets were prepared using a series of applied pressures, and the results were subjected to energy analysis, three dimensional (3D) modeling, and the traditional Heckel analysis. With these approaches, form I was found to be consistently the most brittle material, but the subtle differences between forms II and III were only revealed by 3D modeling. The rank order of the crushing force was found to be I is congruent to II < III. From nanoindentation, form III was found to be much harder than forms I and II, and III also had a much higher Young's modulus. The energy calculations of the nanoindentation curves showed that form III was distinct from forms I and II, which is consistent with the presence of slip planes that are only present in form III. However, in this system, there was little correspondence between the macroscopic and microscopic measurements, and thus particle-particle interactions may to be of paramount importance.

To study the effect of processing conditions on the physical state of mannitol during various stages of the lyophilization cycle of a protein formulation.Mannitol and trehalose were used as the bulking agent and lyoprotectant, respectively. The physical state of mannitol during various stages of freeze-drying cycle, in the absence and presence of a model protein, was characterized using low temperature X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC).Mannitol did not crystallize even when the solution for lyophilization was cooled to -45 degrees C at a slow cooling rate of 1 degree C/min. Annealing facilitated mannitol crystallization, and in the absence of the protein, a mixture of delta-mannitol and mannitol hemihydrate was obtained at both low (-18 degrees C) and high (-8 degrees C) annealing temperatures. However, in the presence of protein, the high annealing temperature promoted delta-mannitol crystallization and inhibited formation of mannitol hemihydrate, while the low annealing temperature facilitated the formation of mannitol hemihydrate. Interestingly, the hemihydrate in the frozen solution was retained in the final lyophile, even when the primary and secondary drying temperatures were as high as -5 and 65 degrees C, respectively.The presence of protein as well as the processing conditions (annealing temperature and time, primary and secondary drying temperatures) influenced the physical form of mannitol in the final lyophile. The protein promoted formation of delta-mannitol while inhibiting the formation of mannitol hemihydrate. Since the physical form of mannitol was greatly influenced by the presence of protein, it will be prudent to conduct the preliminary lyophilization cycle development studies in the presence of the protein. If mannitol hemihydrate is formed during annealing, its dehydration may require high secondary drying temperature.

[Chemical reaction: See text] Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2'-C-beta-difluoromethylribonucleosides. We constructed the glycosylating agent 4 in three steps from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose 1. The key steps included nucleophilic addition of difluoromethyl phenyl sulfone to 2-ketoribose 2 followed by mild and efficient reductive desulfonation. Ribofuranose 4 glycosylated bis(trimethylsilyl)uracil directly, giving difluoromethyluridine 7 efficiently after deprotection. Conversion of 4 to the corresponding ribofuranosyl bromide allowed efficient access to C, A, and G analogues. A related approach starting from methyl D-ribofuranose offered synthetic entry into the diastereomeric manifold, 2'-C-alpha-difluoromethyl-arabino-alpha-pyrimidine. To incorporate 2'-C-beta-difluoromethyluridine into an oligodeoxynucleotide we converted 7 to the bisphosphate and carried out successive ligation reactions using T4 RNA ligase and T4 DNA ligase. Analogous to natural RNA linkages, the resulting oligonucleotide undergoes hydroxide-catalyzed backbone scission at the difluoromethyluridine residue via internal transphosphorylation.

Purpose. High-resolution, tapping-mode atomic force microscopy (AFM) was used to monitor the early stage of the formation of cholesterol crystals under simulated conditions of the gallbladder environment. Methods. AFM images of phosphatidylcholine/cholesterol vesicles were obtained using a mucin-coated mica substrate. Results. The vesicles appeared flattened with diameters from 100 to 300 nm and heights that varied from 10 to 100 nm. Phosphatidylcholine/cholesterol vesicles were mixed with bile salt solutions to yield supersaturated (with respect to cholesterol) dispersions, which were then placed onto mica, silanized mica, and mucin-covered mica substrates. After equilibration, sub-micron sized, plate-like structures were observed at the mica and mucin covered surface, but none were seen at the silanized surface. The morphology was characterized as it pertains to the relative growth rates of the crystal faces. Conclusion. The comparison of these results with literature reports of cholesterol crystals grown in solution suggests that the physical chemical properties of the surface have an important influence in determining the nucleation and subsequent crystal growth of cholesterol.

A new technique developed in this laboratory and based on near-field scanning optical microscopy (NSOM) is used to study the field-induced reorientation of molecules in local regions of thin film materials. A highly concentrated electric field is applied across the sample, between the metal-coated near-field probe and the sample substrate. Molecular motion induced in the sample by modulation of the electric field is observed using NSOM methods. Lock-in detection of the optical response to a sinusoidally- modulated field, recorded under cross-polarized, transmitted-light conditions allows for the recording of dynamics images. The local rate of reorientation is measured for individual locations in a sample by recording the response in either the time or frequency domains. Dynamics information is obtained with microsecond(s) ec time resolution and nanometer-scale spatial resolution. This method is applied in studies of polymer-dispersed liquid crystal films. In these materials, small droplets of nematic liquid crystal are dispersed in an otherwise uniform poly(vinyl alcohol) film. The liquid crystal droplets are birefringent, forming electrically-switchable light-scattering centers. A simple forced-oscillator model for the reorientation dynamics in the liquid crystal is presented. Variations in the time scale and extent of molecular reorientation are observed as a function of field strength, droplet size, droplet shape, and position probed. The data are interpreted based on knowledge of the important intermolecular forces active in these materials.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.