ABSTRACT
Liposomes coated with the poly(amino acid) poly(hydroxyethyl-L-asparagine) (PHEA) show long-circulation properties comparable to the frequently used PEG-liposomes. The pharmacokinetic characteristics of long-circulating liposomes are dependent on the density of the shielding polymer on the liposome surface. Therefore, it is necessary to know the exact composition of the liposomes including the amount of coating polymer present on the liposome surface. In this study, a 1H NMR method to establish the composition of liposomes coated with PHEA was developed and validated.
Subject(s)
Asparagine/analogs & derivatives , Asparagine/chemistry , Liposomes/chemistry , Magnetic Resonance Spectroscopy/methods , Lipids/chemistry , Molecular Structure , Protons , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. C1311 shows significant cytotoxic activity in vitro and in vivo toward a range of colon tumours. The aim of the present study is to develop a sterile and stable, injectable pharmaceutical product for C1311 to be used in phase I clinical trials. C1311 drug substance was structurally and analytically characterised by chromatographic, spectrometric, and diffraction techniques. C1311 was freely soluble in water, and its stability was investigated in several liquid and lyophilised formulations with or without the use of buffering, tonicity, and bulking agents. The final product, containing 100 mg/vial C1311 (as anhydrous free base), was stable for at least 3 months under accelerated storage conditions and at the designated long-term storage condition of 5 +/- 3 degrees C in the dark. The drug is currently used in phase I clinical trials.
Subject(s)
Aminoacridines/chemistry , Antineoplastic Agents/chemistry , Chemistry, Pharmaceutical/methods , Aged, 80 and over , Aminoacridines/administration & dosage , Aminoacridines/analysis , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/analysis , Calorimetry, Differential Scanning/methods , Chromatography, High Pressure Liquid , Clinical Trials, Phase I as Topic , Drug Compounding/methods , Drug Contamination/prevention & control , Drug Stability , Drug Storage/methods , Freeze Drying/methods , Humans , Infusions, Parenteral , Pharmaceutical Solutions , Solubility , Technology, Pharmaceutical/methodsABSTRACT
Liposomes coated with poly(hydroxyethyl L-glutamine) (PHEG) show prolonged circulation times and biodistribution patterns comparable to PEG-coated liposomes. While PEG is a nondegradable polymer, PHEG is expected to be hydrolyzed by proteases. In this study the enzymatic degradability of PHEG both in its free form and grafted onto liposomes was investigated, using the proteases papain, pronase E, and cathepsin B. Enzymatic action was monitored with a ninhydrin assay, which quantifies amine groups formed due to hydrolysis of amide bonds, and the degradation products were characterized by MALDI-ToF mass spectrometry. PHEG, both in its free form and when grafted onto liposomes, showed degradation into low molecular weight peptides by the enzymes. Thus, we present a polymer-coated long-circulating liposome with an enzymatically degradable coating polymer, avoiding the risk of cellular accumulation.
Subject(s)
Enzymes/metabolism , Glutamine/analogs & derivatives , Glutamine/chemistry , Hydrolysis , Liposomes , Magnetic Resonance Spectroscopy , Molecular Weight , Ninhydrin/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The isolation, structure determination, and solution conformation of two conformers of the cyclic heptapeptide phakellistatin 2 (cyclo-[Phe1-cis-Pro2-Ile3-Ile4-cis-Pro5-Tyr6-cis-Pro7]) isolated from the Fijian marine sponge Stylotella aurantium are reported. The conformers can be isolated separately by HPLC and are stable in methanol solution over a period of weeks as determined by NMR. Their NMR spectra and mass spectral fragmentation patterns differ significantly. Their solution conformations were determined by NOE-restrained molecular dynamics calculations and indicated that the two conformers had different folds, hydrogen bonding patterns, and solvent accessible surfaces. These factors may contribute to the independent stability of the two conformers, and may explain the variable biological activity previously reported for phakellistatin 2.
Subject(s)
Peptides, Cyclic/isolation & purification , Porifera/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Drug Screening Assays, Antitumor , Electron Spin Resonance Spectroscopy , Female , Fiji , Humans , Inhibitory Concentration 50 , Leukemia, Myeloid , Ovarian Neoplasms , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Conformation , Stereoisomerism , Tumor Cells, Cultured/drug effectsABSTRACT
Many analytical approaches are available to evaluate (bio)molecular interactions, all of which have their particular advantages and disadvantages. In recent years, two relatively new techniques have emerged that may be used by the bioanalytical community to evaluate such interactions, namely affinity capillary electrophoresis (ACE) and bioaffinity electrospray ionization-mass spectrometry (ESI-MS). In this paper, we describe and evaluate the use of both these techniques for the investigation of the interactions of glycopeptide antibiotics with peptides that mimic the bacterial cell wall binding site. We focus particularly on the effect of the sugar moieties attached to the antibiotic peptide backbone and on the noncovalent dimerization of these glycopeptide antibiotics.
