Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

Structural characterization and solution properties of an acidic branched (1-->3)-beta-D-glucan from Aureobasidium pullulans.

An acidic exopolysaccharide was isolated from a selected strain of Aureobasidium pullulans. On the basis of spectroscopic and chromatographic techniques, the polymer was identified as a beta-D-glucan containing a main chain of (1-->3)-linked beta-D-glucopy-ranosyl units substituted at the O-6 position by single beta-D-glucopyranosyl side chains. The ratio of units in the main chain to units in the side chain was found to be 1.4:1. The ionic character of this exopolysaccharide is due to the presence of malate residues which are linked to the polymer through ester bonds. The degree of substitution was estimated to be very low (0.05). In aqueous solution no signals are present in the NMR spectra strongly suggesting that the polymer adopts a rigid ordered conformation as further confirmed by rheological data. A solvent-induced conformational transition was observed in DMSO in which NMR spectra with good signal-to-noise ratio were obtained. The solution behaviour of the polymer is similar to that of other branched (1-->3)-beta-D-glucans in spite of both the degree of branching and the substitution with malate groups.

Solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae K40.

This paper reports some physicochemical properties of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40 (K40-CPS) in aqueous solution. The polymer has a linear hexasaccharide repeating unit containing one glucuronic acid residue as the only ionizable group. Potentiometric, viscometric, chiro-optical and rheological measurements have been carried out over a range of ionic strength, pH and temperature, with the aim of characterizing the conformational state of the polysaccharide in aqueous solution. All the data reported indicate that the K40-CPS does not undergo a cooperative conformational transition under the investigated experimental conditions. Furthermore, the viscosity data and the viscoelastic spectra suggest that the K40-CPS is rather flexible and adopts a random coil conformation in solution.

Solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae SK1.

The solution properties of the capsular polysaccharide produced by Klebsiella pneumoniae SK1, SK1-CPS, were investigated by various methods. The SK1-CPS repeating unit is a branched pentasaccharide containing one glucuronic acid as single unit side chain; acetyl groups are present as non-carbohydrate substituents on the uronic acid residue in non-stoichiometric amounts. Chiro-optical, potentiometric, viscometric and rheological measurements have been performed in order to characterize the conformational behaviour of the polymer in water and in aqueous salt solutions. Under the investigated experimental conditions, changes of temperature, ionic strength and pH were shown not to induce any cooperative conformational transition. All the results obtained suggest that the solution conformation of SK1-CPS is a random coil with a certain degree of chain flexibility. The removal of the acetyl substituents apparently does not modify the overall conclusions drawn for the native polymer, except for an incipient tendency to aggregation revealed for high salt conditions.

NMR studies of oligosaccharides derived from hyaluronate: complete assignment of 1H and 13C NMR spectra of aqueous di- and tetra-saccharides, and comparison of chemical shifts for oligosaccharides of increasing degree of polymerisation.

A series of oligosaccharides was prepared from hyaluronate by depolymerisation with bovine testicular hyaluronidase. Complete assignment of the 1H and 13C NMR spectra was obtained for the disaccharide, the tetrasaccharide, and the NaBH4-treated tetrasaccharide, by using various 1D and 2D NMR methods. The 1H assignments for the tetrasaccharide differ from the incomplete data reported recently (ref. 11). The 13C NMR spectra of the aqueous di-, tetra-, hexa-, and octa-saccharides of this series show that all resonances, apart from those subject to obvious end effects, have chemical shifts comparable to those of the corresponding resonances of hyaluronate in D2O. The observed 13C chemical shifts suggests that cooperative intramolecular hydrogen bonds probably play a minor role in determining the conformation of hyaluronate in water.

Hyaluronan can be protected from free-radical depolymerisation by 2,6-diisopropylphenol, a novel radical scavenger.

The scavenging effect of 2,6-diisopropylphenol on hydroxy radicals produced by xanthine oxidase was assessed by evaluating the in vitro depolymerisation of hyaluronan in artificial synovial fluid by size-exclusion chromatography. After 1 hour, the number-average molecular weight of hyaluronan remained unchanged (100%) with 2,6-diisopropylphenol, whereas it dropped to 90% with methylprednisolone added, to 55% with the antioxidant 2,6-tert-butyl-4-methylphenol added, and to 10% of its initial value in the absence of scavenger.

Purification and characterization of hyaluronan from synovial fluid.

An easy and rapid method for the purification and characterization of hyaluronan from synovial fluid has been developed. Lipids were removed by filtration through a hydrophobic filter prior to the removal of proteins by phenol-chloroform extraction. The hyaluronan recovery was 95%, as measured by three different methods. The average molecular weight of hyaluronan did not change during the purification. Furthermore, it was found that an optimized enzymatic protein digestion of pathological human synovial fluid prior to filtration yielded up to five times more hyaluronan recovered. In addition, the molecular weight of hyaluronan from synovial fluid not digested with pronase E was apparently higher because of the presence of aggregates. After the purification of hyaluronan (ca. 15 min), a single size-exclusion chromatography step allowed the simultaneous determination of its concentration and the reasonable estimate of its average molecular weight and molecular weight distribution curve. The logarithm of the molecular weight showed a linear dependence on the size-exclusion chromatography elution volume for hyaluronan in the molecular weight range 2.0 x 10(6)-1.0 x 10(4). The removal of proteins allowed the determination of fairly low-molecular-weight fractions of hyaluronan, compared to untreated samples for which hyaluronan and protein peaks partially overlap.