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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 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.

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

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.