Pharmacokinetic and pharmacodynamic properties of an oral formulation of the histone deacetylase inhibitor Belinostat (PXD101).

PURPOSE: The primary objective of this sub-study, undertaken as an extension to the previously reported phase-I study, was to explore the feasibility, tolerability and pharmacokinetics (PK) of belinostat when administered by the oral route. Preliminary pharmacodynamic (PD) studies were also performed to enable comparison of the biological effects of the oral and intravenous formulations. PATIENTS AND METHODS: Oral belinostat was administered in a range of doses and schedules (once, twice or thrice daily), on either day 1 or days 1-5, of the second or a subsequent treatment cycle in 15 patients who were included in the phase-I trial of intravenous belinostat. Serial blood samples were collected for PK and PD (histone acetylation) analyses, and the results compared with corresponding analyses following intravenous administration. RESULTS: A total mean daily AUC of 2,767 ± 1,453 ng h/ml (8.7 ± 4.6 µM h) resulted from a dose of 1,000 mg/m(2) once daily (qd). There was no clear evidence of drug accumulation on twice daily dosing (bid); however, a trend towards accumulation was apparent when belinostat was given three times daily (tid). Mean half-life (T½) of a single dose of 1,000 mg/m(2) was 1.5 h (± 0.3 h) and peak levels were reached in an average of 1.9 h (± 0.3 h). The half-life was found to be independent of dose, but a trend towards increasing half-life following multiple dosing was observed. Histone H4 hyperacetylation in PBMCs estimated after oral dosing was comparable to that achieved after intravenous administration. CONCLUSIONS: High doses of oral belinostat, up to 1,000 mg/m(2) bid for 5 consecutive days, have been tolerated in this small study. An oral formulation could lead to enhanced drug exposure and, more importantly, prolonged effects on the intended drug target. Future trials are required to establish the optimal dose and schedule of oral administration of belinostat.

A phase I study of the safety and pharmacokinetics of the histone deacetylase inhibitor belinostat administered in combination with carboplatin and/or paclitaxel in patients with solid tumours.

BACKGROUND: This phase I study assessed the maximum tolerated dose, dose-limiting toxicity (DLT) and pharmacokinetics of belinostat with carboplatin and paclitaxel and the anti-tumour activity of the combination in solid tumours. METHODS: Cohorts of three to six patients were treated with escalating doses of belinostat administered intravenously once daily, days 1-5 q21 days; on day 3, carboplatin (area under the curve (AUC) 5) and/or paclitaxel (175 mg m(-2)) were administered 2-3 h after the end of the belinostat infusion. RESULTS: In all 23 patients received 600-1000 mg m(-2) per day of belinostat with carboplatin and/or paclitaxel. No DLT was observed. The maximal administered dose of belinostat was 1000 mg m(-2) per day for days 1-5, with paclitaxel (175 mg m(-2)) and carboplatin AUC 5 administered on day 3. Grade III/IV adverse events were (n; %): leucopenia (5; 22%), neutropenia (7; 30%), thrombocytopenia (3; 13%) anaemia (1; 4%), peripheral sensory neuropathy (2; 9%), fatigue (1; 4%), vomiting (1; 4%) and myalgia (1; 4%). The pharmacokinetics of belinostat, paclitaxel and carboplatin were unaltered by the concurrent administration. There were two partial responses (one rectal cancer and one pancreatic cancer). A third patient (mixed mullerian tumour of ovarian origin) showed a complete CA-125 response. In addition, six patients showed a stable disease lasting > or =6 months. CONCLUSION: The combination was well tolerated, with no evidence of pharmacokinetic interaction. Further evaluation of anti-tumour activity is warranted.

Characterization of mixtures of recombinant human cytochrome p450s as a screening model for metabolic stability in drug discovery.

1. Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue. Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery. 2. In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)). Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters. Results were compared with data obtained in human liver microsomes (HLM). 3. Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max). K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold. V(max) differed up to 3-fold between matrices for the CYP forms investigated. However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM. The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged. 4. In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters. Furthermore, addition of antioxidant can in certain cases increase catalytic activity.

Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria.

Tetracyclines used in veterinary therapy invariably will find their way as parent compound and degradation products to the agricultural field. Major degradation products formed due to the limited stability of parent tetracyclines (tetracycline, chlortetracycline, and oxytetracycline) in aqueous solution were theoretically identified at various environmental conditions, such as pH, presence of chelating metals, and light. Their potency was assessed on sludge bacteria, tetracycline-sensitive soil bacteria, and tetracycline-resistant strains. Several of the degradation products had potency at the same concentration level as tetracycline, chlortetracycline, and oxytetracycline on both the sludge and the tetracycline-sensitive soil bacteria. Further, both 5a,6-anhydrotetracycline and 5a,6-anhydrochlortetracycline had potency on tetracycline-resistant bacteria supporting a mode of action different from that of the parent compounds.

Stability of Tylosin A in manure containing test systems determined by high performance liquid chromatography.

Tylosin is a widely used antibiotic for the treatment of infections in swine. Tylosin consists of a mixture of Tylosin A, Tylosin B, Tylosin C and Tylosin D. All components contribute to the potency of tylosin but Tylosin A is by far the major component (usually about 90% and not less than 80%). A fast, robust and easily performed HPLC method has been developed for determination of Tylosin A in the presence of tylosin residues; Tylosin B, Tylosin C and Tylosin D in manure containing incubation media. The separation was performed using a YMC-Pack ODS-AQ column (250 x 4.6 mm i.d., 5 microns particle size) operated at 35 degrees C. The mobile phase consisted of 2.25% (w/v) sodium perchlorate pH 2.5-acetonitrile (60:40 v/v). Detection was performed by measuring the UV absorption at a wavelength of 290 nm. Calibration curves of tylosin made in the incubation medium containing 6.4% manure were linear in the range from 0.375 to 128.0 mg/l (R2 = 0.999). The limit of quantitation (at the RSD 20% level) for Tylosin A was found to be 0.4 mg/l in incubation media containing 6.4% manure. The recovery of Tylosin A was in the range from 100% to 108% depending on the concentration of manure. The reproducibility was good as the relative standard deviation (n = 4) in each matrix tested was in the range from 0.7 to 1.9 at the 25 mg/l level. The stability of Tylosin A was studied under methanogenic conditions and the half-life was found to be less than two days. Studies under aerobic conditions showed that the degradation rate was found to increase with increasing concentrations of manure particles in the incubation medium. It is, however, not clear whether the decrease in the concentration of Tylosin A is caused by sorption, abiotic or biotic chemical degradation. The major degradation product of Tylosin A in methanogenic as well as aerobic incubation media has a UV-spectrum and a retention time corresponding to Tylosin B. Furthermore, Tylosin D is believed to be a minor degradation product.

Non-aqueous capillary electrophoresis of drugs: properties and application of selected solvents.

The electrophoretic mobility of selected acidic and basic test solutes have been determined in non-aqueous media prepared by adding various combinations of ammonium acetate, sodium acetate, methane sulphonic acid and acetic acid to acetonitrile, propylene carbonate, methanol, formamide, N-methylformamide, N,N-dimethylformamide and dimethylsulphoxide, respectively. The apparent pH (pH*) of these non-aqueous media have been measured and it was found that pH* is an important factor for the separations in non-aqueous capillary electrophoresis. However, in some solvents the concentration of sodium acetate has a strong influence on the mobility despite very small changes in pH*. Due to the fact that a change in one parameter influences a number of other parameters it is very difficult to conduct systematic studies in non-aqueous media and to compare the migration of the species at fixed pH* values from one solvent to another. Thus pH* is only of value for comparison when used with a specific solvent or solvent mixture. The viscosity of the above-mentioned solvents were measured at various temperatures and means to adjust the viscosity of the non-aqueous media used for capillary electrophoresis are discussed and the separation of ibuprofen and its major metabolites in urine is used as an example.

Nonaqueous capillary electrophoresis--its applicability in the analysis of food, pharmaceuticals and biological fluids.

The use of nonaqueous electrophoresis media for the application of capillary electrophoresis in the analysis of food, pharmaceuticals and biological fluids is reviewed. Some of the applications are discussed in detail and the benefits of using nonaqueous media in these cases are outlined. Three new applications within pharmaceutical analyses are presented. In these methods either a simple sample pretreatment by dilution with methanol (determination of chlorhexidine in a cream) or selective on-line capillary electrophoresis mass spectrometry (methods for identification of seizure drugs or opium alkaloids) are used. The choice of organic solvents and electrolytes for nonaqueous capillary electrophoresis are discussed. Furthermore, validation data obtained using capillary electrophoresis based on the nonaqueous principle are listed and discussed.

Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis.

A capillary electrophoresis method for determination of the enantiomers of ibuprofen and its major phase I metabolites: 2'-hydroxyibuprofen and 2'-carboxyibuprofen in urine samples have been developed. Cyclodextrins and linear dextrins have been investigated as chiral selectors. Simultaneous chiral separation of the enantiomers of ibuprofen, 2'-hydroxyibuprofen and 2'-carboxyibuprofen was obtained using a mixture of dextrin 10 and heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin in a 2-[N-morpholino]ethanesulphonic acid buffer, pH 5.26. The electroosmotic flow was reversed using hexadimethrine bromide as a buffer additive. The method can be used for the determination of the free enantiomers of ibuprofen, 2'-hydroxyibuprofen and 2'-carboxyibuprofen as well as for the indirect determination of their glucuronic acid conjugates in urine samples.

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids.

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 microg/ml in plasma and 0.14 microg/ml in urine, 1.2 microg/ml for the 4'-glucuronide in plasma and 1.4 microg/ml in urine, and 2.0 microg/ml for the 4-glucuronide in plasma and 3.2 microg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.

Use of metal complexation in non-aqueous capillary electrophoresis systems for the separation and improved detection of tetracyclines.

Metal complexation in non-aqueous capillary electrophoresis systems was evaluated for the separation and improved detection of tetracycline antibiotics using laser-induced fluorescence detection. It was found that three factors were important for the choice of complexing agent: (i) it should be soluble in the organic solvent used for the separation, (ii) it should have a sufficient fast complexing rate so as not to invalidate the electrophoretic separation and, (iii) it should give a large increase in the fluorescence intensity. Mg2+ ions were found to be the most suitable ions for the separation of the tetracyclines as the acetate salt of magnesium is very soluble in organic solvents and only a relatively low current was generated during electrophoresis making it possible to use high concentrations of the complexing metal ion. Metal complexation strongly intensified the fluorescence of tetracyclines and all organic solvents investigated further intensified the fluorescence, e.g. dimethylformamide improved the fluorescence of the oxytetracycline metal complex by a factor of 34 compared to water. However, magnesium acetate was not sufficiently soluble in dimethylformamide and therefore N-methylformamide, improving the fluorescence intensity by only a factor of 9, was used. It was demonstrated that the method can be used for the detection of tetracyclines at the ppb level in milk and plasma.

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica.

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58-86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.

Synthesis, isolation and identification of glucuronides and mercapturic acids of a novel antiparasitic agent, licochalcone A.

1. Four glucuronic acid conjugates of licochalcone A (Lica), and their metabolites, have been synthesized using rabbit and pig liver microsomes and purified by preparative hplc. 2. The glucuronides were identified as E-Lica 4'-O-beta-glucuronide, E and Z-Lica 4-O-beta-glucuronide and a mono-glucuronide conjugate of a beta-hydroxylated Lica metabolite. The metabolites were identified by hplc-nmr (one and two-dimensional nmr) as well as hplc-ms. 3. At pH 8.5 Lica reacted with N-acetyl-L-cysteine giving the two epimeric conjugates, which were then isolated by preparative hplc and identified by one and two-dimensional nmr spectroscopic methods. 4. Only two glucuronic acid conjugates (E- and Z-Lica 4-O-beta-glucuronide) were found in the urine of rat after i.p. administration of a single dose of Lica.

Purification and 1H NMR spectroscopic characterization of phase II metabolites of tolfenamic acid.

Tolfenamic acid, an anti-inflammatory drug (NSAID), is metabolized in vivo to form several oxidative metabolites which are all conjugated with beta-D-glucuronic acid. In this study, the metabolites of tolfenamic acid were identified by 1H nuclear magnetic resonance (NMR) spectroscopy in urine samples obtained on days 7 to 10 from a human volunteer after oral administration of 200 mg of the drug three times per day (steady-state plasma concentration). The metabolites of tolfenamic acid were initially concentrated by preparative solid phase extraction (PSPE) chromatography, thereby removing the endogenous polar compounds that are present in the urine. The individual metabolites were purified by preparative high performance liquid chromatography (HPLC) and then identified using 1H NMR. Both one- and two-dimensional NMR experiments were performed to identify the phase II metabolites of tolfenamic acid; the study shows the applicability of 1H NMR for the identification of drug metabolites in biological fluids. In addition to NMR analysis, two metabolites were also identified by mass spectrometry (MS). The glucuronides of the following parent compounds, N-(2-methyl-3-chlorophenyl)-anthranilic acid (T), N-(2-hydroxymethyl-3-chlorophenyl)-anthranilic acid (1), N-(2-hydroxymethyl-3-chloro-4-hydroxyphenyl)-anthranilic acid (2), N-(2-formyl-3-chlorophenyl) anthranilic acid (3), N-(2-methyl-3-chloro-4-hydroxyphenyl)-anthranilic acid (4), N-(2-methyl-3-chloro-5-hydroxyphenyl)-anthranilic acid (5), N-(2-carboxy-3-chlorophenyl)-anthranilic acid (6), N-(2-hydroxymethyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (7), N-(2-methyl-3-chlorophenyl)-5-hydroxy-anthranilic acid (8), N-(2-methyl-3-chloro-4-metoxyphenyl)-anthranilic acid (9), N-(2-methyl-3-chlorophenyl)-4-hydroxy-anthranilic acid (10), and N-(2-methyl-4-hydroxyphenyl)-anthranilic acid (11) were identified. The phase II metabolites (5-11) had not previously been identified in urine from humans administered tolfenamic acid. The phase I metabolites of the glucuronides 7, 8, 10, and 11 were identified here for the first time. An HPLC method was developed that simultaneously separates all the phase II metabolites identified as well as some phase I metabolites in urine samples obtained after intake of tolfenamic acid.

Validation of a simple method for the determination of oxytetracycline in ointment by non-aqueous capillary electrophoresis.

A non-aqueous capillary electrophoresis method for the determination of oxytetracycline in an ointment have been validated. The oxytetracycline (OTC) is separated from related impurities and degradation products as metal chelates with magnesium ions. Thus, the method show high selectivity. The sample preparation is performed as a single extraction step of OTC from the melted ointment. The test for linearity in the range from 0.2-3.0 mg ml-1 gave a straight line with a coefficient of correlation greater than 0.999. Precision and accuracy were investigated using standard addition at three different concentration levels and six separate determinations at each level. The precision is good and may be expressed as the coefficient of variation which was lesser than 3.6%. The recovery is close to 100%.

A comparative study of precision cut liver slices, hepatocytes, and liver microsomes from the Wistar rat using metronidazole as a model substance.

1. Metronidazole is metabolized by rat liver in vitro models to form a hydroxy metabolite, an acetic acid metabolite, a glucuronic acid conjugate, and a sulphate conjugate. 2. Four different in vitro systems for investigation of drug metabolism based on liver preparations from the male Wistar rat have been investigated. 3. An incubation system where liver slices are incubated in 12-well culture plates was evaluated with respect to metabolism of metronidazole. Optimal viability was observed for a time period of up to 24 h. The Michaelis-Menten parameters for the metabolism of metronidazole in liver slices were calculated and the intrinsic clearance values compared with the values determined in hepatocytes incubated in suspension. It was found that the intrinsic clearance with respect to formation of oxidative metabolites, the hydroxy metabolite, and the acetic acid metabolite correlated, whereas the intrinsic clearance with respect to formation of the glucuronic acid conjugate was lower in slices compared with hepatocytes. 4. The metabolism of metronidazole in liver slices, in hepatocytes in primary monolayer culture, in hepatocytes incubated in suspension, and in liver microsomes was compared. All the incubations were performed under identical incubation conditions including the same incubation medium. The trend observed was that the initial metabolic rates of the production of the hydroxy metabolite, the glucuronic acid metabolite, and the acetic acid metabolite of metronidazole were higher in microsomes than in the other liver preparations. The metabolic rates in hepatocytes in primary culture and in suspension with respect to the oxidative metabolites were higher than in liver slices. The metabolic turnover observed in liver slices was predicted to correlate with in vivo data earlier obtained for rat.

Nonaqueous capillary electrophoresis in pharmaceutical analysis.

Nonaqueous media have shown to be useful for CE separations. It is possible to obtain high selectivities of closely related compounds without using any additive in the electrophoresis medium. The physicochemical parameters of some selected solvents and their influence on separation are discussed. Separations of small compounds with equal or almost equal charge-to-mass ratios have been performed in both aqueous- and nonaqueous-based media and compared. Examples of the use of nonaqueous media for drug impurity testing are given, and precautions for obtaining quantitative and reproducible analysis in nonaqueous media are discussed.

Separation of basic drug substances of very similar structure using micellar electrokinetic chromatography.

Small molecules having very similar molecular structure--some even with the same mass over charge--are not trivial to separate by capillary electrophoresis in free solution. However, addition of surfactants to the electrophoretic buffer and thus using the principle of micellar electrokinetic chromatography may provide separation with high resolution. The use of zwitterionic and nonionic surfactants is demonstrated and an example of how a developed system may be used for drug purity testing is given.

Identification of oxidation products of 5-aminosalicylic acid in faeces and the study of their formation in vitro.

The formation of three oxidant-derived products of 5-aminosalicylic acid (5-ASA) in vivo was demonstrated in patients with active ulcerative colitis as well as in healthy subjects. The products were isolated from faeces by preparative HPLC and their chemical structures were found to be oxidation products of 5-ASA using 1H-NMR spectroscopy and mass spectrometry. Reactions carried out in vitro between 5-ASA and oxidants suggested to be present in the inflamed bowel verified that the hypochlorite-mediated oxidation of 5-ASA as well as the haemoglobin-catalysed H2O2-dependent oxidation of 5-ASA resulted in the formation of a single oxidation product of 5-ASA. This product was similar to, but not identical to any of the products identified in faeces from patients receiving 5-ASA. Oxygen radical-mediated oxidation of 5-ASA gave several products, different from the products isolated. Finally, it was verified that the products formed in vivo are not formed as a result of autooxidation of 5-ASA either in faeces extract or in pharmaceuticals.

High-performance liquid chromatographic assay of 5-aminosalicylic acid (5-ASA) and its metabolites N-beta-D-glucopyranosyl-5-ASA, N-acetyl-5-ASA, N-formyl-5-ASA and N-butyryl-5-ASA in biological fluids.

A fast, highly sensitive high-performance liquid chromatographic method for the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its metabolites N-beta-D-glucopyranosyl-5-ASA, N-formyl-5-ASA, N-acetyl-5-ASA and N-butyryl-5-ASA has been developed using a dynamically modified silica approach on a 40 mm x 4.6 mm I.D. column packed with 3-microns Hypersil. Plasma proteins are precipitated with acetonitrile. After extraction of the acetonitrile into 1,1,1-trichloroethane an undiluted aqueous phase containing the analytes is obtained. The detection limits are in the range 0.002-0.05 microgram/ml in plasma at a signal-to-noise ratio of 3 using fluorescence detection.

Stability of 5-aminosalicylic acid and its metabolites in plasma at -20 degrees C. Formation of N-beta-D-glucopyranosyl-5-aminosalicylic acid.

The stability of 5-aminosalicylic acid and its metabolites has been investigated when stored frozen. N-beta-D-Glucopyranosyl-5-aminosalicylic acid was formed in considerable amounts concomitant with a decrease in 5-aminosalicylic acid in plasma samples spiked with 5-aminosalicylic acid as well as in standard solutions of 5-aminosalicylic acid buffered with potassium phosphate between pH 5.5 and pH 8.0 with 4.0 mM glucose added and stored at -20 degrees C. Thus N-beta-D-glucopyranosyl-5-aminosalicylic acid might not, as previously described, be a metabolite of 5-aminosalicylic acid but an artifact formed during storage of plasma samples. The N-glucoside formed could be quantitatively degraded to 5-aminosalicylic acid and glucose by adding 0.2 M potassium phosphate buffer pH 3.0 to the sample prior to the analysis. The metabolites of 5-aminosalicylic acid (N-formyl-5-aminosalicylic acid, N-acetyl-5-aminosalicylic acid and N-butyryl-5-aminosalicylic acid) were found to be stable in plasma stored at -20 degrees C for at least eight months.