Use of X-ray crystallography for the characterization of single crystals grown in steroid containing transdermal drug delivery systems.

Target of the study was to characterize crystals which had grown in steroid-containing matrix patches during short-term storage and to thereby establish a rationale for the inhibition of crystal formation in those patches in general. Matrix type transdermal drug delivery systems (TDDS) containing either 2.2% gestodene or 3.3% estradiol were free of crystals directly after their production. However, crystals of up to 800 microns in length grew during 3 months of storage at ambient temperature. The application of several analytical methods did not help to identify the crystals. This was mainly due to the fact that the adhesive matrix surrounding the crystals could not be fully removed in the course of sample preparation with routine laboratory methods and thus impaired DSC, FTIR microscopy and hot stage polarized microscopy. However, within X-ray diffractometry, the residual amorphous patch matrix did not hamper the measurement of the crystals. Thus, they were identified as estradiol hemihydrate and gestodene form I, respectively. These results suggest that steroid-containing matrix TDDS should be stabilized against drug recrystallization e.g. by the addition of suitable crystallization inhibitors. Furthermore, systems containing estradiol may be stabilized by efficient removal water.

Synthesis and Physicochemical Characterization of a New Gadolinium Chelate: The Liver-Specific Magnetic Resonance Imaging Contrast Agent Gd-EOB-DTPA.

A convenient synthesis of disodium S-[4-(4-ethoxybenzyl)-3,6,9-tris[(carboxy-kappaO)methyl]-3,6,9-triazaundecandioato)(5-)-kappa(3)N(3)(),N(6)(),N(9)(),kappa(2)O(1)(),O(11)()]gadolinate(2-) (Gd-EOB-DTPA), 1, is reported. This water-soluble complex is presently undergoing phase III clinical trials as a liver-specific contrast agent for magnetic resonance imaging (MRI). The thermodynamic complex stability constant of 1 and the acid dissociation constants of the ligand have been determined as well as the stability constant of the calcium complex Ca-EOB-DTPA (2), which is used as an additive in the pharmaceutical formulation of the contrast agent. The solid-state structure of the ligand S-4-(4-ethoxybenzyl)-3,6,9-tris(carboxylatomethyl)-3,6,9-triazaundecanedioic acid (H(5)EOB-DTPA), 3, has been elucidated in a single crystal X-ray diffraction study. Additionally, HPLC evidence is given that the enantiomerically pure ligand forms two diastereomeric gadolinium complexes in a 65:35 ratio. The kinetics of isomerization of the isolated diastereomers-as dependent on pH and temperature-has been investigated, and thus, the activation energy for the interconversion of these isomers has been estimated to be 75.3 kJ mol(-1). Finally, the structures of the two components of 1 are discussed in terms of four possible diastereomers.

Synthesis and Structure of a New Macrocyclic Polyhydroxylated Gadolinium Chelate Used as a Contrast Agent for Magnetic Resonance Imaging.

Three approaches to the synthesis of a new ligand 1,4,7-tris(carboxymethyl)-10-(1-(hydroxymethyl)-2,3-dihydroxypropyl)-1,4,7,10-tetraazacyclododecane (6) are described. This ligand forms the both thermodynamically and kinetically very stable gadolinium chelate Gadobutrol (1), which is a neutral and highly hydrophilic compound that is used for magnetic resonance imaging in the clinic. According to the crystal structure the Gd(III) ion in 1 is nine coordinated. The ligand provides eight coordination sites whereas the ninth coordination partner surprisingly is a carboxylate oxygen of a neighboring centrosymmetrically-related complex molecule. Ligand 6 was also utilized to prepare the calcium complex 12 which is used as an additive in the pharmaceutical formulation of 1. For the calcium complex 12, two complex molecules adopting almost identical conformations are present in the crystal.

Cloning, purification, and crystallization of Escherichia coli cystathionine beta-lyase.

The metC gene coding for cystathionine beta-lyase of Escherichia coli has been cloned and used to construct an overproducing E. coli strain. An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent. The crystals belong to the orthorombic space group C222. Their unit cell parameters are a = 60.9 A, b = 154.7 A and c = 152.7 A. Consideration of the possible values of VM accounts for the presence of one dimer per asymmetric unit. The crystals are suitable for X-ray analysis and a complete native date set to 1.83 A resolution has been collected using synchrotron radiation.

Three-dimensional structure of fatty-acid-binding protein from bovine heart.

The complex of fatty-acid-binding protein (FABP) from bovine heart (cFABP, pI4.9) with endogenous lipid was crystallized in the presence of ammonium sulfate as precipitant. The needle-shaped crystals belong to the monoclinic space group C2, with unit-cell constants a = 5.262(6) nm, b = 7.631(8) nm, c = 3.945(5) nm and beta = 94.47(9) degrees. A native data set to 0.35 nm resolution was collected using synchrotron radiation and film methods. An initial model for the three-dimensional structure of the protein was constructed based on the crystal structure of the related bovine P2 myelin protein [Jones, T.A., Bergfors, T., Sedzik, J. & Unge, T. (1988) EMBO J. 7, 1597-1604] to which the amino acid sequence of bovine cFABP was adapted. Energy minimizations were carried out under different conditions using both an all-atom and a united-atom force field. The optimized models were used to determine the crystal structure of cFABP by molecular-replacement techniques. The structure was refined by simulated annealing to R = 0.267. As the bound lipid is heterogeneous, it could not be located in the electron-density map and/or the attained resolution was not sufficient. Bovine cFABP is composed of ten antiparallel beta strands forming a beta barrel, and by two alpha helices. The structural features are similar to those of other members of the superfamily of hydrophobic molecule transporters.

The three-dimensional structure of the bifunctional proteinase K/alpha-amylase inhibitor from wheat (PK13) at 2.5 A resolution.

Wheat germ contains an inhibitor for proteinase K, called PK13 (Mr approximately 19,600) which simultaneously inhibits alpha-amylase. PK13 was crystallized, space group P21, a = 43.02 (5) A, b = 65.18 (7) A, c = 32.33 (4) A, beta = 112.79 degrees (9), X-ray data were collected to 2.5 A resolution, the structure solved by molecular replacement on the basis of the atomic coordinates of the homologous Erythrina caffra DE-3 inhibitor, and refined with simulated annealing techniques with a current R-factor of 21%. The three-dimensional structure of PK13 is stabilised by two disulfide bridges and has a central beta-barrel with distorted beta-structure. In analogy to related inhibitors, the binding site for proteinase K is assumed to be located on the surface of the protein (amino acid residues 66-67), although the 75-76 peptide bond is cleaved upon binding.

Crystal structure of a zwitterionic detergent: n-octyl-2-hydroxyethylsulfoxide.

The crystal structure of n-octyl-2-hydroxyethylsulfoxide (1) (space group P2(1)/c, a = 16.516(7), b = 9.053(4), c = 8.222(4) A, beta = 97.58 degrees) was determined by X-ray diffraction methods to 0.94 A resolution and refined to R = 0.050 (Rw = 0.052). In the crystal lattice the molecules are not arranged tail-to-tail, as usually observed with amphiphilic compounds, but head-to-tail and aligned in the a-axis direction. They form layers in the b, c plane which are interdigitated such that adjacent molecules in one layer are in antiparallel orientation. The packing is stabilized by intermolecular hydrogen bonds O-H--O-S. No solvent molecules were detectable.

Rapid preparation of the nicotinic acetylcholine receptor for crystallization in detergent solution.

A novel rapid purification method for the nicotinic acetylcholine receptor from Torpedo electric tissue was developed. It allows preparation of 10 mg quantities of pure and stabile receptor protein within 2 days. This protein is used for crystallization attempts. Conditions are described which reproducibly yield crystals.