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.

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.

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.

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography.

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70 degrees C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C8 column with acetonitrile-0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10,000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 microl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at -30 degrees C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1,000 mg/m2 CP in a 1-h infusion demonstrated the applicability of the assay.

Pharmaceutical development of a parenteral lyophilized formulation of the novel antitumor agent aplidine.

Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was characterized by using several chromatographic and spectrometric techniques. These experiments showed that aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-drying was carried out with a 500 micrograms/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection (WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to determine the optimal freeze-drying cycle parameters. The prototype, containing 500 micrograms aplidine and 25 mg D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both reconstituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formulation is stable for at least 1 year when stored at +2-8 degrees C in the dark.

Pharmacokinetics and metabolism of the staurosporine analogue CGP 41 251 in mice.

Studies with CGP 41 251 (I), an N-benzoylstaurosporine derivative and PKC-alpha inhibitor, revealed that oral administration of 400 microg/day of the compound to wild type mice on four successive days reversed multi drug resistance (Killion et al. Oncology Research 7: 453-459, 1995). In our study, the same regimen of administration was followed with the primary objective to establish the pharmacokinetics and metabolism of the compound and to substantiate at which plasma concentrations of CGP 41 251 multi drug resistance (MDR) reversal can be expected. Concentrations of CGP 41 251 and metabolites in plasma were determined by a validated high performance liquid chromatography (HPLC) method with fluorescence detection. Structural characterization of the metabolites was performed with HPLC and mass spectrometric detection. In our experiment extensive metabolism of CGP 41 251 was found. The presence of five hydroxylated metabolites of CGP 41 251 (I) was confirmed and two metabolites were structurally elucidated as CGP 50 750 (III) and CGP 52 421 (V). Maximal concentrations of 73 ng/ml, 1.9 ng/ml and 126 ng/ml for CGP 41 251 (I), III and V were found, respectively. The mass spectra of the other three metabolites indicate that these are oxidized nitrogens or hydroxylated compounds. As yet, the oxidation or hydroxylation sites have not been established. This study has revealed new information about CGP 41 251 pharmacokinetics and metabolism. Target levels between 10-100 ng/ml may be important to achieve in further clinical trials with CGP 41 251 as MDR modulator.

A search for new metabolites of N,N',N''-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.

Degradation study of the investigational anticancer drug clanfenur.

Clanfenur belongs to a new group of substituted benzoylphenylureas. The drug shows both in vitro and in vivo antitumour activity. To assess its chemical stability, a study was carried out in which the effect of pH, temperature, ionic strength and buffer concentration on the reaction rate constant k(obs) were examined. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) system was used. The pH-log k(obs) degradation profile, obtained at 70 degrees C, shows that clanfenur has its maximum stability in the pH region 4-5. At pH 7, half-lives were calculated by extrapolation of the Arrhenius plot; at 4 degrees C the half-life was calculated to be 141 years and at 25 degrees C 9. 5 years. The activation energy was calculated to be 114 kJ/mol. In acidic, neutral, and alkaline media, the ionic strength has no effect on the degradation. The buffer concentration of citrate, phosphate, borate, and carbonate did not affect the value of k(obs). An RP-HPLC chromatogram of degraded clanfenur shows the presence of four degradation products, three of which were identified by LC-ESI-MS as p-chloroaniline, p-chlorophenylurea and 2-fluoro-6-dimethylaminobenzamide.

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4-hydrazino-2-stilbazole.

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole (4'H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis-trans isomerism at the proline-pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4'H2S is performed in a water-acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4'H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational antitumor neuropeptide antagonist [Arg6, D-Trp7,9, MePhe8]-Substance P [6-11].

The aim of this study was to develop a stable parenteral dosage form for the investigational cytotoxic drug [Arg6, D-Trp79,MePhe8]-Substance P [6-11] (Substance P Antagonist G; Antagonist G). Antagonist G bulk drug was structurally and analytically characterized. The drug exhibits excellent aqueous solubility, although relatively poor aqueous stability characteristics. Lyophilization was, therefore, selected as the manufacturing process. Differential scanning calorimetry studies were conducted to determine the freeze-drying cycle parameters which resulted in a stable, lyophilized formulation of Antagonist G. The prototype, containing 50 mg Antagonist G per vial, was found to be the optimal formulation in terms of solubility, length of the freeze-drying cycle, stability, and dosage requirements in the planned phase I clinical trials. Quality control of the freeze-dried formulation showed that the manufacturing process does not change the integrity of Antagonist G. Shelf life studies demonstrated that the formulation is stable for at least 3 years, when stored at 2-8 degrees C in a dark environment. Oxidative degradation products of Antagonist G were isolated and structurally characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, and infrared spectroscopy.

High-performance liquid chromatographic determination of stabilized 4-hydroxyifosfamide in human plasma and erythrocytes.

A method using reversed-phase high-performance liquid chromatography (RP-HPLC) is described for the measurement of the stabilized activated metabolite of ifosfamide, 4-hydroxyifosfamide (4-OHIF), in human plasma and erythrocytes. Immediately after sample collection and plasma-erythrocyte separation at 4 degrees C, 4-OHIF was stabilized by derivatization with semicarbazide (SCZ). The sample pretreatment involved liquid-liquid extraction with ethyl acetate. RP-HPLC was executed with a C8 column and acetonitrile-0.025 M potassium dihydrogenphosphate buffer (pH 7.40)-triethylamine (13.5:86:0.5, v/v) as mobile phase. The analyte was determined with UV detection at 230 nm. Complete validation, optimisation and stability studies were performed and the method proved to be specific, sensitive and with a stable analyte in the range of clinically relevant concentrations (0.1-10 microg/ml) after conventional dosing. The lower limit of quantitation was 100 ng/ml using 1.00 ml of sample. Accuracy was between 94.1 and 107.0%. Within-day and between-day precisions were less than 6.2% and 7.2%, respectively. 4-OHIF-SCZ was found to be stable in the biological matrix at -20 degrees C for at least 1 month. A pharmacokinetic study conducted in a patient receiving 9 g/m2 over 3 days by means of a continuous infusion, demonstrated the applicability of this method.

A novel pyrroleninone cross-link from bovine dentine.

The aim was to identify suspect collagen cross-links in dentine, eluting close to known cross-links in ion-exchange HPLC. Bovine tooth roots as source of dentine were powdered, demineralised, reduced, and acid-hydrolysed. Cross-linking amino acids were isolated from the acid hydrolysate by size exclusion, adsorption, and sequential ion exchange chromatography. In addition to dihydroxylysinonorleucine and hydroxylysylpyridinoline, an unknown cross-link was isolated (V-2). The ultraviolet, mass, and nuclear magnetic resonance spectra support the proposed structure of V-2, a trimeric amino acid with a pyrroleninone nucleus.

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.

Pharmaceutical development of a parenteral formulation of the investigational anticancer drug clanfenur.

A stable parenteral dosage form for the investigational cytotoxic drug clanfenur was designed, and the bulk drug was characterized by its nuclear magnetic resonance, mass spectrometry, infrared, and ultraviolet spectra. The 1H and 13C spectra show clanfenur to be a mixture of two stereoisomers. Because of poor solubility in aqueous solution and precipitation in co-solvent, surfactant, or emulsion systems, a two-pump infusion system was developed for intravenous administration. Clanfenur, solubilized in a Cremophor EL/ethanol (1:1, w/v) solution (concentration, 15 mg/mL), can be simultaneously infused with 5% dextrose infusion fluid. Total doses of up to 1,680 mg of clanfenur (and 56 g of Cremophor EL) theoretically can be administered to patients over a 6-hour period. From accelerated stability testing of clanfenur in the Cremophor EL/ethanol (1:1, w/v) formulation, a shelf life of 3.5 years at 4 degrees C and of 4 months at 25 degrees C is calculated.

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

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 detected 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.

Podacycline A and B, two cyclic peptides in the latex of Jatropha podagrica.

Two novel cyclic peptides were isolated from the latex of Jatropha podagrica, which we named podacycline A and B. Podacycline A is a cyclic nonapeptide with the sequence Gly1-Leu2-Leu3-Gly4-Ala5-Val6-Trp7-Ala8-Gly9+ ++-Gly1. The sequence of podacycline B, a cyclic heptapeptide, was determined to be Phe1-Ala2-Gly3-Thr4-Ile5-Phe6-Gly7-Phe1. The amino acid residues of both compounds were found to have the L-configuration.

Cyclogossine A: a novel cyclic heptapeptide isolated from the latex of Jatropha gossypifolia.

From the latex of Jatropha gossypifolia L. (Euphorbiaceae) a novel cyclic heptapeptide was isolated, which we named cyclogossine A. A combination of amino acid analysis, FAB mass spectrometry, and two dimensional 1H-NMR spectroscopy (TOCSY and ROESY) was used to determine the primary structure. The compound was found to contain one glycine, one alanine, one valine, two leucine, one threonine, and one tryptophan residue; its amino acid sequence is: Leu 1 - Ala 2 - Thr 3 - Trp 4 - Leu 5 - Gly 6 - Val 7. The absolute configurations of the amino acids were determined by chiral gas chromatography; all have the L-configuration.

Purity determinations in the bulk drug of the novel indoloquinone antitumor agent EO9.

The pharmaceutical development of the investigational cytotoxic drug EO9 included the structural characterization of the bulk drug by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and infrared (IR) spectroscopy, and analytical characterization by high-performance liquid chromatography and ultraviolet/visible spectrophotometry. The presence of impurities in the bulk drug was investigated. The intermediates in the synthesis of EO9 were structurally characterized by NMR spectroscopy and MS, and analytically characterized by HPLC analysis with photodiode array (PDA) detection. All of the intermediates were below their limits of detection in EO9 bulk drug. The amounts of residual organic solvents were determined by gas chromatography. Methanol and ethanol were detected, but the amounts present did not exceed the limits as set in the United States Pharmacopeia XXII.