Pharmacokinetics of anticancer drugs in vitro.

It is generally assumed that drug concentration does not change significantly under cell culture conditions. Nevertheless, most of the therapeutic trials in acute leukemia that were based on in vitro drug sensitivity assays of patient samples have been disappointing. In order to show possible pitfalls of unphysiological alterations in vitro we investigated concentration versus time curves, metabolism and effects on the culture media for some antineoplastic drugs. Oxazaphosphorines and cytarabine were incubated in RPMI and in established cell lines and measured by HPLC. HPLC also served to measure enzyme activity and levels of related amino acids at various concentrations of asparaginase, ammonia release was photometrically determined. Etoposide was monitored by HPLC relative to different contents of FCS in RPMI. All oxazaphosphorines showed a rapid decrease of in vitro activity down to about 10% within 4-6 h, and 2% within 72 h. The level of cytarabine, when incubated in RPMI, was stable over 24h, and no change was seen with K562, while a rapid decrease to below 50% occurred within 6h in the presence of HL 60 and BLIN. 2 U/L of asparaginase led to asparagine depletion of the medium within 4h, while 200 U/L were associated with a preferential increase of glutamic acid and ammonia. Further, there was evidence of instability by rapid adsorption to plastic surfaces (paclitaxel) or isomerisation (etoposide) in RPMI with low FCS content. The instability of drugs in vitro is attributed to a variety of different factors: i.e. physico-chemical instability results in inactivation of oxazaphosphorines, cytarabine disappears by cellular metabolism without saturation depending on the cell-line. Epiphenomena like adsorption and isomerisation in vitro are unphysiological. Results of drug sensitivity assays should be interpreted with great caution.

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.

Analysis of the components of Lycopus europaeus L. in body fluids during metabolism studies. Comparison of capillary electrophoresis and high-performance liquid chromatography.

During pharmacokinetic studies with extracts obtained from medicinally used plants, analysis in body fluids is mainly performed by HPLC, an established separation method. In this paper high-performance capillary electrophoresis (HPCE) is investigated for its ability to separate such complex extracts. Crude extracts of Lycopus europaeus L. (Lamiaceae) are traditionally used against mild forms of hyperthyroidism. The metabolism of a 70% ethanolic extract with respect to some of its individual main components (rosmarinic and caffeic acid, luteolin-7-glucoside) and a mixture of the pure compounds were investigated using isolated perfused rat liver. After solid-phase extraction metabolites were determined using HPCE and HPLC separation techniques. A buffer solution composed of 0.05 mol l-1 Na2HPO4 at pH 7.0 with 30% acetonitrile was found to be the most suitable electrolyte for HPCE separation. The best mobile phase for isocratic HPLC was 0.03% TFA-acetonitrile (82:18, v/v). Data obtained with HPCE are in good accordance with those from HPLC; HPCE, however, is clearly more rapid and simple to perform.

The effect of Chelidonium majus herb extract on choleresis in the isolated perfused rat liver.

The total ethanolic extract, the phenolic and the alkaloidal fraction of the herb of Chelidonium majus L. (Papaveraceae) were tested for their choleretic activity using the isolated perfused rat liver. The total extract significantly caused chloresis by increasing the bile acid independent flow (BAIF); the observed weak activity of both fractions, tested each and as combination, however, was not significant.

Endocrine effects of Lycopus europaeus L. following oral application.

Lycopus extracts are used in folk medicine for the treatment of hyperthyroid symptoms. Diverse effects on the pituitary thyroidal system as well as on the pituitary gonadal system could be confirmed in experimental studies. But till now endocrine effects of Lycopus extracts in experimental animals were observed after parenteral application only. Therefore in this investigation an ethanolic extract of Lycopus europaeus was applied orally to rats, diverse endocrine parameters were measured between 3 and 24 h later and the effects compared to an i.p. treated group. The plant extract given p.o. caused a long lasting (for a period of more than 24 h) decrease of T3 levels, presumably as a consequence of a reduced peripheral T4 deiodination. A pronounced reduction of T4 and thyroid stimulating hormone (TSH) concentrations was observed 24 h after application of the test solution by gavage. The luteinizing hormone (LH) decrease as well as the TSH decrease, which was pronounced in spite of reduced T4 and T3 levels indicate a central point of attack of the plant extract. Differences in the biological activity in dependence on the route of application may be explained e.g. by differences in absorption of plant constituents.

Metabolism of caffeic acid in the isolated perfused rat liver.

Caffeic acid as well as its oxidation products exert a spectrum of biological effects in in vitro testing. To get an idea of the amount as well as the mode of biotransformation, caffeic acid metabolism was investigated by means of the isolated perfused rat liver. The first-pass effect was not pronounced: 93.3% of caffeic acid dose appeared unchanged after one liver passage. Products of caffeic acid oxidation (cyclolignan derivatives) as well as ferulic and isoferulic acid as methylation products were found in the perfusion medium. In addition, a cyclization product, esculetin, was observed. In the bile, mainly glucuronides as well as sulfates of caffeic acid could be determined. Thus, oxidation products and other metabolites formed by liver metabolism can be responsible for the biological effects in vivo.

The oxidation of caffeic acid derivatives as model reaction for the formation of potent gonadotropin inhibitors in plant extracts.

Synthetic caffeic acid derivatives, substoichiometrically oxidized with KMnO4, exhibit antigonadotropic activity against pregnant mare serum gonadotropin (PMSG) to a greater degree than caffeic acid itself. Inhibitory compounds, formed after an oxidation of caffeic acid and its derivatives are bound to PMSG dependent on their concentration to result in hormone-inhibitor complexes. These PMSG-inhibitor complexes exhibited little or no biological activity, depending on the structure of the inhibitor. The substoichiometric oxidation with KMnO4 led to the corresponding unstable o-quinones as first products. The complete oxidation reaction could be divided into an initial KMnO4-dependent step followed by a manganese-catalyzed autoxidation, which was accompanied by a pronounced oxygen uptake from the solution. The HPLC analysis after an oxidation of caffeic acid derivatives led to product patterns with strong similarities to those of caffeic acid in the respective product UV spectra, suggesting the formation of compounds with similar structures.

The NSAID sulindac reverses rectal adenomas in colectomized patients with familial adenomatous polyposis: clinical results of a dose-finding study on rectal sulindac administration.

After colectomy with ileorectal anastomosis (IRA) for treatment of familial adenomatous polyposis (FAP), the rectal mucosa remains, with the risk of malignant change. Locoregional (rectal) sulindac has been applied, with initial higher-dose therapy and subsequent low-dose maintenance therapy to minimise side-effects. The dose-finding study with sulindac suppositories started with a dose of 300 mg sulindac daily per patient over 6 weeks. Depending on proctoscopical evaluation of regression of polyposis, sulindac doses were reduced in predefined steps. Ten of 15 patients developed a complete remission following 42 weeks of treatment, while the rest had partial remission. Responses were recorded 6-24 weeks after beginning sulindac treatment. After 36 weeks, 13/15 patients received 25-50 mg sulindac daily. An increase in the number of partial remissions after 42 weeks of treatment at doses of 100 mg sulindac daily may indicate the first approach to a reduced dose between 100 mg to 25 mg sulindac daily, but may also point to the importance of long-term treatment rather than dose-intense therapy.

On the influence of the sugar moiety on the antigonadotropic activity of luteoline glycosides.

Luteolin-7-glucoside and luteolin-7-glucuronide were isolated from Lycopus europaeus L. (Lamiaceae) and identified by 1H- and 13C-NMR spectroscopy. Luteolin-7-glucuronide proved to be active against PMSG in vitro as well as in vivo, whereas the other flavone glycosides occurring in the plant were completely inactive.

Structures of compounds with antigonadotropic activity obtained by in vitro oxidation of caffeic acid.

Two new cyclolignan derivatives were isolated by HPLC from the mixture of substances obtained after oxidation of caffeic acid with KMnO4. Their structures were elucidated by spectroscopic methods as 2,3-dicarboxy-6,7-dihydroxy-1-(3', 4'-dihydroxy)-phenyl-1, 2-dihydronaphthalene (1) and 3-carboxy-6,7-dihydroxy-1-(3', 4'-dihydroxy)-phenylnaphthalene (2). Compounds 1 and 2 exhibit antigonadotropic activity as do the extracts of crude drugs of Lycopus europaeus and Lithospermum officinale after oxidation by plant enzymes.

Oxidation products of caffeic acid as model substances for the antigonadotropic activity of plant extracts.

Phenolic plant constituents exert antigonadotropic activity following an oxidation. The resulting complex mixture of mostly instable products impedes the elucidation of the various oxidation steps as well as the mode of antigonadotropic action. Thus caffeic acid was chosen as a single model phenolic to facilitate the interpretation. The oxidation of caffeic acid with KMnO4 as well as with polyphenoloxidase leads to the instable caffeic acid o-quinone as the first oxidation product. Following the initial oxidation, a number of products was indicated via HPLC. Two of them were isolated and characterized as oligomers of caffeic acid, one of them with phenolic, acid and, quinoic structural components and a relative molecular mass similar to caffeic acid tetramer. It was shown that caffeic acid quinone cannot be the antigonadotropically active principle. Correspondingly, the isolated oxidation products exhibit pronounced antigonadotropic activity. It could be proved that oxidation products of caffeic acid bind to PMSG, forming PMSG-inhibitor-complexes. In such complexes the gonadotropic activity of PMSG is completely abolished.

Formation of compounds with antigonadotropic activity from inactive phenolic precursors.

The antigonadotropic activity of Lithospermum and Lycopus species can be attributed to their phenolic components. These compounds represent precursors of biologically active products which are formed by an oxidation step. Complexity and instability of these products aggravates the elucidation of detailed structural properties. Therefore, the type of reaction involved had to be clarified. Among the oxidation products of phenolic substances, the corresponding quinones are found. It can be demonstrated that the reaction between quinones and unoxidized diphenols yields products with strong antigonadotropic activity. This type of reaction - the formation of quinhydrones - is proposed to be engaged in the formation of various products with antigonadotropic activity.