Pharmacokinetics of flubendazole and its metabolites in lambs and adult sheep (Ovis aries).

Flubendazole (FLU) is indicated for control of helminthoses in pig and avian species (monogastric animals) and its corresponding pharmacokinetics are well known. The information on FLU's pharmacokinetic behavior in animal species with forestomach (ruminants) has been limited although the use of FLU in these species could be beneficial. The aim of this study was to investigate the pharmacokinetics of FLU and its main metabolites in sheep. The effects of animal age (sexually immature and mature ones) and gender were also studied. FLU was orally administered in a single experimental dose (30 mg/kg of body weight) in the form of oral suspension. Treated immature animals (aged 3 months) and 5 months later the same mature individuals (aged 8 months) were kept under the same conditions (food, water and management) and treated with FLU. Within 72 h after FLU administration, plasmatic samples were collected and FLU and its Phase I metabolites were quantified using high-performance liquid chromatography. FLU was detected in very low concentrations only, reduced FLU (FLU-R) was identified as the main metabolite, and hydrolyzed FLU (FLU-H) as the minor one. Formation of FLU-R was stereospecific with (+)-FLU-R domination. The plasmatic concentrations of (+)-FLU-R reached 10-15 times higher values than those of FLU, (-)-FLU-R and FLU-H. A significant gender effect on pharmacokinetics of FLU or (+)-FLU-R metabolite in the mature animals was found and a wide significant difference between lambs and adult sheep in FLU including both metabolites has been proved.

High-performance liquid chromatography-tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites.

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002-2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H](+) and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H](-) ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS( n ) analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.

Biotransformation of flubendazole and selected model xenobiotics in Haemonchus contortus.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g., sheep and goat). The treatment of haemonchosis is complicated because of frequent resistance of H. contortus to common anthelmintics. The development of resistance can be facilitated by the action of drug metabolizing enzymes of parasites that can deactivate anthelmintics and thus protect parasites against the toxic effect of the drug. The aim of this project was to investigate the Phase I biotransformation of benzimidazole anthelmintic flubendazole in H. contortus and to determine the biotransformation of other model xenobiotics. For this purpose, in vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (live nematodes cultivated in flasks with medium) experiments were used. The results showed that cytosolic NADPH-dependent enzymes of H. contortus metabolize flubendazole via reduction of its carbonyl group. The apparent kinetic parameters of this reaction were determined (V'max=39.8+/-2.1 nM min(-1), K'm=1.5+/-0.3 microM). The reduction of flubendazole in H. contortus is stereospecific, the ratio of (-):(+) enantiomers of reduced flubendazole formed was 90:10. Reduced flubendazole was the only Phase I metabolite found. Effective reduction of other xenobiotics with carbonyl group (metyrapon, daunorubicin, and oracin) was also found. Significant activity of carbonyl-reducing enzymes may be important for H. contortus to survive the attacks of anthelmintics or other xenobiotics with carbonyl group.

Miotic action of tramadol is determined by CYP2D6 genotype.

Polymorphic CYP2D6 is the enzyme that activates the opioid analgesic tramadol by O-demethylation to its active metabolite O-demethyltramadol (M1). Our objective was to determine the opioid effects measured by pupillary response to tramadol of CYP2D6 genotyped volunteers in relation to the disposition of tramadol and M1 in plasma. Tramadol displayed phenotypic pharmacokinetics and it was possible to identify poor metabolizers (PM) with >99% confidence from the metabolic ratio (MR) in a single blood sample taken between 2.5 and 24 h post-dose. Homozygous extensive metabolizers (EM) differed from PM subjects by an almost threefold greater (P=0.0014) maximal pupillary constriction (Emax). Significant correlations between the AUC and Cmax values of M1 versus pupillary constriction were found. The corresponding correlations of pharmacokinetic parameters for tramadol itself were weaker and negative. The strongest correlations were for the single-point metabolic ratios at all sampling intervals versus the effects, with rs ranging from 0.85 to 0.89 (p<0.01). It is concluded that the concept of dual opioid/non-opioid action of the drug, though considerably stronger in EMs, is valid for both EM and PM subjects. This is the theoretical basis for the frequent use and satisfactory efficacy of tramadol in clinical practice when given to genetically non-selected population.

High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma.

Mesalazine (5-aminosalicylic acid, 5-ASA), an anti-inflammatory agent for the treatment of inflammatory bowel diseases, is metabolized in organism to the principal biotransformation product, N-acetyl-5-ASA. Some other phase II metabolites (N-formyl-5-ASA, N-butyryl-5-ASA, N-beta-d-glucopyranosyl-5-ASA) have also been described. 5-ASA is a polar compound and besides it exhibits amphoteric properties. The extraction of this compound from biomatrices and its chromatographic analysis is complicated. In order to improve the reliability of the determination of parent 5-ASA, a derivatization of 5-ASA together with 4-ASA (added to samples as a precursor of I.S.-2) was involved into the method. More lipophilic N-propionyl-5-ASA and N-propionyl-4-ASA (I.S.-2) were obtained using propionic anhydride. These derivatives were well extractable together with N-acyl-5-ASAs (metabolites) and N-acetyl-4-ASA (I.S.-1). As the first internal standard (I.S.-1) was used for the evaluation of extracted N-acyl-metabolites, the second internal standard (I.S.-2) served for the evaluation of both derivatization and extraction steps of parent drug 5-ASA. Based on these reasonings, new HPLC bioanalytical method for the determination of 5-ASA and its metabolites in blood plasma was developed and validated. The sample preparation step consists of the deproteination of plasma by HClO(4) and the above-mentioned derivatization of ASAs followed by liquid-liquid extraction of all N-acyl-ASA-derivatives. Chromatographic analyses were performed on a 250-4 mm column containing Purospher RP-18 e, 5 microm (Merck, Darmstadt, Germany) with a precolumn (4-4 mm). The column effluent was monitored using both UV photodiode-array (lambda = 313 nm) and fluorescence detectors (lambda(exc.) = 300 nm/lambda(emiss.) = 406 nm) in tandem. The identity of individual N-acyl-ASAs in the extracts from biomatrices was verified by characteristic UV-spectra and by HPLC/MS experiments. The whole analysis lasted 23 min at the flow rate of 1 ml min(-1). LLOQ (LOD) was estimated 126 (20) pmol ml(-1) of plasma for N-acetyl-5-ASA and 318 (50) pmol ml(-1) of plasma for N-propionyl-5-ASA. The validated HPLC method was applied to pharmacokinetic studies of mesalazine in humans and animals.

High-performance liquid chromatographic determination of ciprofloxacin in plasma samples.

A new bioanalytical high-performance liquid chromatographic (HPLC) method for the determination of ciprofloxacin with norfloxacin as an internal standard was developed and validated for plasma samples. Norfloxacin is structural homologue of ciprofloxacin and exhibits similar retention properties. The quality of respective peak separation is strongly influenced by amphoteric character of ciprofloxacin and norfloxacin as well. In previously published HPLC methods on conventional C18 reversed-phase [F. Belal, A.A. Al-Majed, A.M. Al-Obaid, Talanta 50 (1999) 765-786; G. Carlucci, J. Chromatogr. A 812 (1998) 343-367], ion pair reagents were added into the mobile phase to suppress peak tailing. In comparison with endcapped and high purity silica reversed-phase sorbent (Purospher RP-18e, Merck), which yielded symmetrical peaks, separation efficiency was further enhanced in our method. Gradient elution mode using acetonitrile and phosphate buffer pH 3 on the pentafluorophenylpropyl stationary phase (250-4.6 mm Discovery HS F5, 5 microm, Supelco) was carried out. The resolution of 4.1 for ciprofloxacin-norfloxacin peaks was achieved. Sample preparation by SPE C18 (Supelclean) with recovery 72% was performed. Fluorescence detection with lambda(excit)=280 nm, lambda(emis)=446 nm was used. After the validation, the bioanalytical HPLC method was applied to pharmacokinetic studies.

Use of chiral liquid chromatography for the evaluation of stereospecificity in the carbonyl reduction of potential benzo[c]fluorene antineoplastics benfluron and dimefluron in various species.

Benfluron (B) [5-(2-dimethylaminoethoxy)-7H-benzo[c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway (O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm x 4.6 mm ID, 5 microm) using a mobile phase acetonitrile-1 M NaClO4 (40:60, v/v). Another mobile phase, methanol-1 M NaClO4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min(-1) in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.

Disposition study of a new potential antineoplastic agent dimefluron in rats using high-performance liquid chromatography with ultraviolet and mass spectrometric detection.

The disposition of a new potential antineoplastic drug dimefluron after an oral administration to rats was investigated. Dimefluron, 3,9-dimethoxy-5-(2-dimethylaminoethoxy)-7H-benzo[c]fluoren-7-one hydrochloride, was administered in a single oral dose (250 mg kg(-1) of body weight) in the form of an aqueous solution via a gastric probe. Dimefluron metabolites were being searched for in rat faeces. Synthetic standards of the expected phase I metabolites (the products of O- and N-desmethylation, N-oxidation and carbonyl reduction of dimefluron) were prepared and used together with dimefluron and internal standard in the development of two HPLC bioanalytical methods based on different separation principles. The first separation of dimefluron and the phase I metabolites was tested on a 250 mm x 4 mm chromatographic column with LiChrospher 60 RP-selectB 5 microm (Merck) using an isocratic mobile phase containing 0.01 M nonylamine buffer (pH 7.4) and acetonitrile in the 1:2 ratio (v/v). The second separation was performed on a 250 mm x 4 mm chromatographic column Discovery HS F5, 5 microm (Supelco) using a linear gradient mode with the mobile phase containing acetonitrile and phosphate buffer (0.05 M KH2PO4, pH 3). The flow rate was 1 ml min(-1) in both cases. UV detection was performed in the dual wavelength mode, with 317 nm having been used for dimefluron and all 7H-benzo[c]fluoren-7-one metabolites, 367 nm for 7H-benzo[c]fluoren-7-ol metabolites. A higher homologue of dimefluron served as an internal standard. The identity of the dimefluron metabolites in biological samples was confirmed using HPLC-MS experiments. The elimination study showed that the concentration maximum for dimefluron and its metabolites in rat faeces was reached 48 h after the administration of the parent drug. O-Desmethylated derivatives of dimefluron prevailed among the phase I metabolites.

Identification and determination of phase II nabumetone metabolites by high-performance liquid chromatography with photodiode array and mass spectrometric detection.

Chromatographic analyses play an important role in the identification and determination of phase I and phase II drug metabolites. While the chemical standards of phase I metabolites are usually available from commercial sources or by various synthetic, degradation or isolation methods, the phase II drug metabolites have usually more complicated structures, their standards are in general inaccessible and their identification and determination require a comprehensive analytical approach involving the use of xenobiochemical methods and the employment of hyphenated analytical techniques. In this work, various high-performance liquid chromatography (HPLC) methods were employed in the evaluation of xenobiochemical experiments leading to the identification and determination of phase II nabumetone metabolites. Optimal conditions for the quantitative enzymatic deconjugation of phase II metabolites were found for the samples of minipig bile, small intestine contents and urine. Comparative HPLC analyses of the samples of above-mentioned biomatrices and of the same biomatrices after their enzymatic treatment using beta-glucuronidase and arylsulfatase afforded the qualitative and quantitative information about phase II nabumetone metabolites. Hereby, three principal phase II nabumetone metabolites (ether glucuronides) were discovered in minipig's body fluids and their structures were confirmed using liquid chromatography (LC)-electrospray ionization mass spectrometric (MS) analyses.

Comparative biotransformation and disposition studies of nabumetone in humans and minipigs using high-performance liquid chromatography with ultraviolet, fluorescence and mass spectrometric detection.

The disposition of the non-steroidal anti-inflammatory drug (NSAID) nabumetone after a single oral dose administration of nabumetone tablets to humans and minipigs was investigated. Nabumetone is a prodrug, which is metabolized in the organism to the principal pharmacodynamically active metabolite -- 6-methoxy-2-naphthylacetic acid (6-MNA), and some other minor metabolites (carbonyl group reduction products, O-desmethylation products and their conjugates with glucuronic and sulphuric acids). Standards of the above-mentioned metabolites were prepared using simple synthetic procedures and their structures were confirmed by NMR and mass spectrometry. A simple HPLC method for the simultaneous determination of nabumetone, 6-MNA and the other metabolites was developed, validated and used for xenobiochemical and pharmacokinetic studies in humans and minipigs and for distribution studies in minipigs. Naproxen was chosen as the internal standard (I.S.), both UV (for higher concentrations) and fluorescence detection (for very low concentrations) were used. The identity of the nabumetone metabolites in biological samples was confirmed using HPLC-MS experiments. Pharmacokinetics of nabumetone, 6-MNA and 6-HNA (6-hydroxy-2-naphthylacetic acid) in human and minipig plasma was evaluated and compared. The concentration levels of nabumetone metabolites in urine, bile and synovial fluid were also evaluated.

High-performance liquid chromatographic determination of tramadol and its O-desmethylated metabolite in blood plasma. Application to a bioequivalence study in humans.

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.

Evaluation of cardiac effects of the new antineoplastic drug --dimethoxybenfluron--in the rabbit.

Cardiotoxicity ranks among the most serious adverse effects of some cytostatics. The cardiac effects of repeated i.v. administration of a new antineoplastic agent, dimethoxybenfluron (once a week, 10 administrations), were investigated in rabbits with respect to cardiac function and the release of cardiac troponin T (cTnT). Different doses of dimethoxybenfluron were administered to two groups of animals (12 mg/kg; n = 7 and 24 mg/kg; n = 6) and compared with either a control group (saline 1 ml/kg; n = 6) or a group with experimentally induced cardiomyopathy (daunorubicin 50 mg/m2; n = 13). In daunorubicin-induced cardiomyopathy, cTnT levels in animals with premature deaths were significantly higher (0.31 +/-0.11 microg/l) in comparison with the surviving animals (0.04 +/- 0.03 microg/l). However, cardiac TnT levels after the administration of dimethoxybenfluron in both doses were within the physiological range (lower than 0.1 microg/l) during the whole experiment as it was in the control group. The lack of cardiotoxicity of this new antineoplastic drug was supported by the absence of alterations in PEP:LVET ratio, left ventricle dP/dtmax or histological heart examination as well as by the fact that no premature death of animals occurred following repeated administration of dimethoxybenfluron. It is possible to conclude that no signs of cardiotoxicity were observed following repeated i.v. administration of dimethoxybenfluron.

High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization.

Ursodeoxycholic acid (3 alpha,7 beta-dihydroxy-5 beta-cholanoic acid, UDCA) is a therapeutically applicable bile acid widely used for the dissolution of cholesterol-rich gallstones and in the treatment of chronic liver diseases associated with cholestasis. UDCA is more hydrophilic and less toxic than another therapeutically valuable bile acid, chenodeoxycholic acid (CDCA), the 7 alpha-epimer of UDCA. Procedures for sample preparation and HPLC determination of UDCA in blood serum were developed and validated. A higher homologue of UDCA containing an additional methylene group in the side chain was synthetized and used as an internal standard (IS). Serum samples with IS were diluted with a buffer (pH=7). The bile acids and IS were captured using solid phase extraction (C18 cartridges). The carboxylic group of the analytes was derivatized using 2-bromo-2'-acetonaphthone (a detection-oriented derivatization), and reaction mixtures were analyzed (HPLC with UV 245 nm detection; a 125--4 mm column containing Lichrospher 100 C18, 5 microm; mobile phase: acetonitrile--water, 6:4 (v/v)). Following validation, this method was used for pharmacokinetic studies of UDCA in humans.

[Plasma levels of troponin T in rabbits after administration of new antineoplastic agents (Oracin and Dimefluron) and daunorubicin]. / Plazmatické hladiny troponinu T u králíku po podání nových antineoplastických látek (Oracinu a Dimefluronu) s daunorubicinem.

Recently, cardiac troponin T (cTnT) has been shown to be a sensitive marker of anthracycline-induced cardiomyopathy. In our study, the cardiotoxicity of repeated i.v. administration (once a week, 10 administrations) of daunorubicin combined with new antineoplastic drugs (with mild side-effects) were followed in two groups of rabbits: 1) Dimefluron (3,9-dimethoxybenfluron hydrochloride-12 mg/kg) + daunorubicin (3 mg/kg), 2) Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H- indeno[1,2c]isoquinoline hydrochloride--10 mg/kg) + daunorubicin (3 mg/kg) and compared with the control group (saline--1 ml/kg) and the group with experimentally induced cardiomyopathy (daunorubicin--3 mg/kg). The concentration of cTnT in heparinized plasma samples was measured using commercial kit (Roche). In the control group, plasma levels of cTnT were always within the physiological range (i.e. lower than 0.1 microgram/l) during the experiment. During the development of daunorubicin-induced cardiomyopathy, after the eighth administration of drug, cTnT was significantly higher (0.31 +/- 0.11 microgram/l) in animals with premature deaths compared with the rest of the group (0.04 +/- 0.03 microgram/l). The animals with pathological values of cTnT were at higher risk of premature deaths (P = 0.0006). The combination of daunorubicin either with Oracin or with Dimefluron caused neither significant changes of cTnT levels nor significant deterioration of other followed-up parameters (especially, functional and toxicological parameters). Similarly to the daunorubicin group, the animals with pathological levels of cTnT after the eighth administration of antineoplastic drugs were at higher risk of premature death (P = 0.025). Our results show that the plasma concentration of cardiac troponin T could be a suitable predictive marker of cardiotoxicity of antineoplastic drugs.

Effect of substituents on microsomal reduction of benzo(c)fluorene N-oxides.

The potential benzo(c)fluorene antineoplastic agent benfluron (B) displays high activity against a broad spectrum of experimental tumours in vitro and in vivo. In order to suppress some of its undesirable properties, its structure has been modified. Benfluron N-oxide (B N-oxide) is one of benfluron derivatives tested. The main metabolic pathway of B N-oxide is its reduction to tertiary amine B. A key role of cytochrome P4502B and P4502E1 in B N-oxide reduction has been proposed in the rat. Surprisingly, B N-oxide is reduced also in the presence of oxygen although all other N-oxides undergo reduction only under anaerobic conditions. With the aim to determine the influence of the N-oxide chemical structure and its redox potential on reductase affinity, activity and oxygen sensitivity five relative benzo(c)fluorene N-oxides were prepared. A correlation between the redox potential measured and the non-enzymatic reduction ability of the substrate was found, but no effect of the redox potential on reductase activity was observed. Microsomal reductases display a high affinity to B N-oxide (apparent K(m) congruent with0. 2 mM). A modification of the side-chain or nitrogen substituents has led to only a little change in apparent K(m) values, but a methoxy group substitution on the benzo(c)fluorene moiety induced a significant K(m) increase (ten-fold). Based on kinetic study results, the scheme of mechanism of cytochrome P450 mediated benzo(c)fluorene N-oxides reduction have been proposed. All benzo(c)fluorene N-oxides under study were able to be reduced in the presence of oxygen. Changes in the B N-oxide structure caused an extent of anaerobic conditions preference. The relationship between the benzo(c)fluorene N-oxide structure and the profile of metabolites in microsomal incubation was studied and important differences in the formation of individual N-oxide metabolites were found.

[Effects of new antineoplastic agents (dimethoxybenflurone and Oracin) on the cardiovascular system and other parameters in the rabbit in vivo]. / Ucinky nových antineoplastických látek (dimethoxybenfluronu a Oracinu) na kardiovaskulární systém a dalsí parametry u králíku in vivo.

Anthracycline derivatives belong among the most effective antineoplastic drugs but their therapeutic use is limited by their side effects--a dose-related cardiotoxicity. The influence of repeated i.v. administration (once weekly, max. 10 weeks) of new antineoplastic agents--dimethoxybenfluron (DMB) (3,9-dimethoxybenfluron hydrochloride, C23H24O4NCl, M.w. 413.9, Institute of Experimental Biopharmaceutics, Czech Academy of Sciences, Hradec Králové, Czech Republic; 12 or 24 mg base/kg) and Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H- indeno[1,2c]isoquinoline hydrochloride), C20H19N2O3Cl, M.w. 370.84, Research Institute for Pharmacy and Biochemistry, Prague, Czech Republic; 5 or 10 mg/kg) on cardiovascular, biochemical, haematological and histological parameters were studied in rabbits in vivo. Data obtained in these groups were compared with the group with experimentally induced cardiomyopathy (daunorubicin 50 mg/m2 i.v.) and with the control group (saline 1 ml/kg). Only mild and mostly no significant changes of the cardiovascular parameters (DMB 12 group: PEP:LVET ratio--0.408-0.502, LV dP/dtmax.--1337.0 kPa/s; DMB 24 group: PEP:LVET ratio--0.407-0.433, LV dP/dtmax.--1438.2 kPa/s), biochemical parameters (decrease in natrium, ALP and increase in glucose, GPX and GSH levels) and haematological parameters (increase in erythrocytes and decrease in leukocytes after the larger dose of the drug) were found in the dimethoxybenfluron groups. Repeated administration of the lower dose of Oracin induced only mild and mostly no significant changes of parameters (PEP:LVET ratio--0.393-0.475, LV dP/dtmax.--1092.4 kPa/s) in comparison with the control group. Though significant in some intervals, only a mild oscillation of the PEP:LVET ratio (0.368-0.446), decrease in LV dP/dtmax. (991.2 kPa/s) and--in comparison with control group--significantly higher blood pressure and lower heart rate were found after the higher dose of Oracin. In the most of haematological and biochemical parameters (with the exception of chlorides, protein and albumin levels) no significant changes were present. Histological examination of the heart revealed normal structure of the myocardium including minute changes of myocardium following administration of antineoplastic agents in all groups. Administration of new antineoplastic agents induced mostly mild changes of the followed-up parameters (PEP:LVET ratio, LV dP/dtmax., heart rate, levels of cardiac troponin T, survival of animals, haematological and biochemical parameters); the values of parameters were mostly significantly different from those in rabbits with daunorubicin-induced cardiomyopathy. On the basis of our results it is possible to conclude that the administration of dimethoxybenflurone and Oracin did not induce signs of cardiotoxicity in rabbits in vivo. This observation is considered to be important from the viewpoint of possible further clinical use of these new antineoplastic agents.

Study of the biotransformation of benfluron using the isolated perfused rat liver.

The isolated perfused rat liver method (IPRL) was used to find, isolate and identify further metabolites of Phase I and Phase II biotransformation of the potential cytostatic agent benfluron with special regard to the conjugation processes. Its pharmacokinetic profile during the perfusion was also estimated. The rat liver was isolated from the body and perfused in vitro using a recirculating perfusion system. Benfluron was added to the reservoir as a bolus in doses of 200, 100, 30 mg/kg of body weigh and 1 mg/perfusate volume and also as a continual infusion in a dose of 0.1 mg/min in separate series of experiments. The following metabolites formed during Phase I biotransformation were found in the perfusion liquid as well as in the bile: benfluron N-oxide, 9-hydroxy benfluron, demethylated 9-hydroxy benfluron, demethylated benfluron, and reduced benfluron. The major Phase II metabolite found in the bile samples was the glucuronide of 9-hydroxy benfluron. The pharmacokinetic profile of benfluron in IPRL indicated its main disposition and metabolic pathway, i.e. its rapid extraction from perfusate by the liver (t1/2 alpha = 3.76 min), 9-hydroxylation followed up O-glucuronidation and excretion to the bile. It was revealed that 12% of the total dose of the parent compound was excreted to the bile in the form of conjugates during the first hour of perfusion, 32% during 1.5 hour, and 70% during 2 hours after the administration of benfluron. The conjugates with glucuronic acid represented 96-98% of all metabolites found in the bile.

The influence of dimethoxybenfluron on biochemical and haematological parameters in rabbits.

The influence of repeated i.v. administration of dimethoxybenfluron (NO-1-B) (12 or 24 mg base/kg once weekly, 10 weeks) on biochemical and haematological parameters were studied in rabbits in vivo. No significant changes were mostly found in the serum ion levels between the dimethoxybenfluron and the control groups, as well as in most of other biochemical parameters (including total protein and albumin levels). Nevertheless, the lower dose of dimethoxybenfluron caused an increase in the glucose level. Furthermore, no significant changes were mostly present also in haematological parameters in the dimethoxybenfluron groups of rabbits (a mild decrease in thrombocytes and leucocytes). The results of our study support an assumption of good tolerance of dimethoxybenfluron from the viewpoint of its influence on biochemical and haematological parameters in rabbits and may be considered of importance for a possible therapeutic use of the derivatives.

Experimental Goettingen minipig and beagle dog as two species used in bioequivalence studies for clinical pharmacology (5-aminosalicylic acid and atenolol as model drugs).

Due to proven similarities in biotransformation between man and minipig, minipig seems to be the experimental animal of choice for preclinical pharmacokinetic studies when an experiment with a drug exhibiting a great first pass bioelimination (like 5-aminosalicylic acid) is to be realised. On the other hand, both minipig and dog may be suitable species for a pharmacokinetic study with a drug characterized by a small extent of first pass biotransformation (like atenolol).

Distribution of fenofibric acid in lipoprotein fractions of patients.

The antidyslipidemic agent fenofibrate (procetofen) is hydrolysed in vivo to its main active metabolite--fenofibric (procetofenic) acid. This metabolite is usually determined in pharmacokinetic studies, because plasma concentrations of fenofibrate are practically undetectable. Presented study is focussed on the distribution of fenofibric acid into lipoprotein (VLDL, LDL, IDL and HDL) fractions of human and (for comparison) minipig blood plasma, which has not been studied yet. In order to obtain more accurate results, a new HPLC method based on the use of newly synthetized internal standards was developed. Four homologues of fenofibric acid prepared have identical chromophoric part of their molecules and hence the same UV spectra as fenofibric acid. From this point of view, these standards are more suitable for determination of fenofibric acid than the formerly used ones--naproxen or bezafibrate. Fenofibric acid levels in the high density lipoprotein fraction has been shown to be significantly higher (in both human and minipig plasma) than in the other lipoprotein fractions. This fact may be explained by higher affinity of the fenofibric acid to proteins constituting major part of the high density lipoprotein fraction.