J. J. Kettenes-van den Bosch

New insights into the hydrolytic degradation of poly(lactic acid): participation of the alcohol terminus

Abstract The hydrolytic degradation of monodisperse lactic acid oligomers was studied in vitro to gain insight into the degradation of oligolactic acid grafted to dextran, which we use for the preparation of hydrogels based on physical interactions, or the degradation of PLA/PLGA. The decrease in the amount of oligomer and the formation of degradation products was monitored by HPLC and MS. The amount of lactic acid oligomer decreased according to pseudo-first-order kinetics and was dependent on the dielectric constant of the medium and the pH. The OH end group was found to play a crucial role in the hydrolytic degradation; when the OH was blocked no significant degradation was observed. At acidic pH, hydrolysis was shown to proceed by chain-end scission whereas in alkaline medium, lactoyl lactate was split off. The possible consequences of these findings for the degradation of PLA matrices are discussed.

Physically crosslinked dextran hydrogels by stereocomplex formation of lactic acid oligomers: degradation and protein release behavior

Hydrogels, physically crosslinked through stereocomplex formation, were obtained by mixing aqueous solutions of dextran with L-lactic acid grafts and dextran with D-lactic acid grafts. Protein-loaded hydrogels were simply prepared by dissolving the protein in these dextran solutions prior to mixing. It was shown that under physiological conditions the gels are fully degradable. When the gels were exposed to an aqueous buffer solution, they first showed a swelling phase in which their weight increased 2-3 times due to absorption of water, followed by a dissolution phase. The degradation time depended on the composition of the hydrogel, i.e., the number of lactate grafts, the length and polydispersity of the grafts and the initial water content, and varied from 1 to 7 days. Most likely, the degradation of the stereocomplex hydrogel started with hydrolysis of the carbonate ester, which links the lactate graft to dextran. The gels showed a release of the entrapped model proteins (IgG and lysozyme) over 6 days and the kinetics depended on the gel characteristics, such as the polydispersity of the lactate grafts and the initial water content. Lysozyme was mainly released by Fickian diffusion, indicating that its hydrodynamic diameter is smaller than the hydrogel mesh size. On the other hand the release of IgG was governed by diffusion as well as swelling/degradation of the hydrogel. Importantly, the proteins were quantitatively released from the gels and with full preservation of the enzymatic activity of lysozyme, emphasizing the protein-friendly preparation method of the protein-loaded stereocomplex hydrogel.

Curcacycline A — a novel cyclic octapeptide isolated from the latex of Jatropha curcas L.

From the latex of Jatropha curcas L. (Euphorbiaceae) a novel cyclic octapeptide was isolated, which we named curcacycline A. The compound was found to contain one threonine, one valine, two glycine, and four leucine residues. By two‐dimensional 1H‐NMR spectroscopy (HOHAHA and ROESY), its sequence was determined to be Gly1‐Leu2‐Leu3‐Gly4‐Thr5‐Val6‐Leu7Leu8‐Gly1. Curcacycline A displays a moderate inhibition of (i) classical pathway activity of human complement and (ii) proliferation of human T‐cells.

Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran.

A novel hydrogel system in which crosslinking is established by stereocomplex formation between lactic acid oligomers of opposite chirality is proposed. To investigate the feasibility of this novel system, we first investigate whether there is an operation window where lactic acid oligomers in either the D- or L-form do not give a crystalline phase, whereas in a blend of the D- and L-form stereocomplex formation occurs. Therefore, D- and L-lactic acid oligomers with different degrees of polymerization (DP) were prepared and analyzed using DSC. It was shown that crystallinity was present in D- or L-oligomers with DP > or = 11. On the other hand, in blends of D- and L-oligomers of lactic acid crystallinity (stereocomplexation) was already observed at a DP > or = 7. In the next step, L- and D-lactic acid oligomers were coupled via their terminal hydroxyl group to dextran, yielding dex-(L)lactate and dex-(D)lactate, respectively. Upon dissolving each product in water separately and mixing the solutions, a hydrogel is formed at room temperature as demonstrated by rheological measurements. The storage modulus of the obtained hydrogel strongly decreased upon heating to 80 degrees C, while it was restored upon cooling to 20 degrees C demonstrating the thermo-reversibility and the physical nature of the cross-links. The storage modulus of the gels depends on the degree of polymerization of the lactate acid grafts and their degree of substitution on dextran. Interestingly, gel formation was favored when one lactic oligomer was coupled via its hydroxyl group whereas the oligomer of opposite chirality was coupled via its carboxylic acid group. This is ascribed to the parallel packing of the oligomers in stereocomplexes.

Gas chromatographic/mass spectrometric assay for profiling the enantiomers of 3,4-methylenedioxymethamphetamine and its chiral metabolites using positive chemical ionization ion trap mass spectrometry

A qualitative GC/MS profile was obtained and its mass spectrometric features characterized for the analysis of the enantiomers of (RS)-3,4-methylenedioxymethamphetamine (MDMA) and its metabolites (RS)-3,4-methylenedioxyamphetamine (MDA), (RS)-4-hydroxy-3-methoxymethamphetamine (HMMA) and (RS)-4-hydroxy-3-methoxyamphetamine (HMA). A chiral derivatization method was selected to obtain the diastereomers required for the separation of the respective enantiomers with a non-chiral GC stationary phase. The selected derivatization consisted of a reaction with N-heptafluorobutyryl-(S)-prolyl chloride combined with a consecutive reaction with N-methyl-N-trimethylsilyltrifluoroacetamide, resulting in N-[heptafluorobutyryl-(S)-prolyl]-O-trimethylsilyl derivatives. Detection was carried out with electron ionization and positive chemical ionization (PCI) ion trap mass spectrometry. Mass spectra of the derivatives of reference standards of the compounds of interest obtained with PCI demonstrated that this method simultaneously induces proton and charge-transfer reactions in the ion trap. The advantage is that high mass information is provided while some fragmentation remains to elucidate structural details. Subsequently, in three urine samples obtained from different and unrelated MDMA intoxications, the enantiomers of MDMA and MDA were identified. In some urine samples also HMMA and/or HMA were found. In addition to these compounds, an unexpected compound and/or additional chiral metabolite, N-hydroxy-(RS)-3,4-methylenedioxyamphetamine, was identified in two out of three urine samples. Preliminary results also indicated an enantioselective metabolism in the N-demethylation pathway for MDMA in humans.

Isolation and structural confirmation of N-desmethyl topotecan, a metabolite of topotecan

Abstract A sensitive high-performance liquid chromatography (HPLC) method for the determination of topotecan and total levels of topotecan (lactone plus its ring-opened hydroxycarboxylate form) was developed by the authors and used in several pharmacokinetics studies. During the analysis of plasma and urine samples collected in those studies, an additional peak eluting just after topotecan was observed. Approximately 100 ng of this potential metabolite was isolated from human urine using a solid-phase extraction procedure and purification by HPLC. Analysis of the isolated material by HPLC showed it to be approximately 95% pure. Mass spectrometry data along with the HPLC retention data and fluorescence data (in comparison with synthetic reference standard) are consistent with the metabolite’s being N-desmethyl topotecan. The maximal concentrations of metabolite deteced in human plasma and urine were relatively low. When topotecan was given as a 30-min infusion at 1.0 mg/m2 daily for 5 days every 3 weeks, the maximal plasma metabolite concentration (lactone plus the ring-opened hydroxycarboxylate form) was about 0.7% (n=4) of the maximal total topotecan concentration. The average amount of metabolite excreted in urine during the treatment was 1–4% (n=20) of the delivered dose.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF THE STABILIZED CYCLOPHOSPHAMIDE METABOLITE 4-HYDROXYCYCLOPHOSPHAMIDE IN PLASMA AND RED BLOOD CELLS

A reversed-phase high performance liquid chromatographic (HPLC) method for the determination of the activated cyclophosphamide (CP) metabolite 4-hydroxycyclophosphamide (4-OHCP) in human plasma and red blood cells is described. 4-OHCP is very unstable in biological matrices. Therefore, it was treated, immediately after sampling, with semicarbazide to form a stable semicarbazone derivative, which was identified with electron spray mass spectrometry. The derivative was analysed with HPLC with ultraviolet (UV) detection at 230 nm. Sample pre-treatment consisted of a liquid-liquid extraction with ethyl acetate, the chromatography system was a 25 cm C8 column (particle size 5 μm) with a mobile phase of acetonitrile-0.025 M potassium phosphate buffer (15:85 v/v) pH 6.0. After assay optimisation, the method was validated and stability studies were carried out. The method proved linear in clinically relevant concentrations (50-5000 ng/mL). The lower limit of quantitation was 50 ng/mL. Accuracy and precision were below 7% over the full concentration range. A calibration curve in plasma can also be used for the quantification of 4-OHCP in red blood cells. The derivative was found to be stable for at least 11 months when stored at −70 °C. A pharmacokinetic pilot study in a patient treated with 1000 mg/m2 CP in combination with thioTEPA and carboplatin demonstrated the applicability of the assay for the determination of 4-OHCP in plasma and red blood cells.

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.

Chemical hydrolysis of DOTAP and DOPE in a liposomal environment

In this study the hydrolysis kinetics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyltrimethylammoniumpropane (DOTAP) in net neutral DPPC-DOPE (3:1, mol/mol) and cationic DOTAP-DOPE (1:1, mol/mol) liposomes are described. The log k(obs)-pH profile for DOTAP-DOPE liposomes differs markedly from earlier observed hydrolysis profiles: the slope approaches zero in the acidic region and +1 in the alkaline region. The concept of amine-influenced hydrolysis is introduced to explain the lack of pH dependency in the acidic region of the log k(obs)-pH profiles.

HPLC-UV METHOD DEVELOPMENT AND IMPURITY PROFILING OF THE MARINE ANTICANCER AGENT APLIDINE IN RAW DRUG SUBSTANCE AND PHARMACEUTICAL DOSAGE FORM

The development and validation of a reversed-phase high performance liquid chromatographic (RP-HPLC) method with ultra-violet (UV) detection for the quantification and purity determination of aplidine in raw drug substance and pharmaceutical dosage form is described. Using this method, the aplidine rotamers present as a consequence of cis-trans isomerism of the peptide bond between the proline and pyruvoyl moieties in the molecule, elute as one single peak. Linear calibration curves in the range of 12.5–300 μg/mL of aplidine with correlation coefficients > 0.999 were obtained. Within-run and between-run precisions were ≤ 2.2% and accuracy was within 100.6–101.4%. A profile of recurrent impurities was drawn up from several lots of aplidine raw drug substance manufactured thus far. Major impurities were identified as didemnin-A, acetyldidemnin A, and noraplidine using liquid chromatography-mass spectrometry (LC-MS). No significant differences in chromatographic profile between aplidine raw drug substance and its pharmaceutical dosage form were found.