Phase I and pharmacological study of daily oral administration of perifosine (D-21266) in patients with advanced solid tumours.

Alkylphosphocholines are a novel class of antitumour agents structurally related to ether lipids that interact with the cell membrane and influence intracellular growth signal transduction pathways. We performed a phase I trial with an analogue of miltefosine, perifosine (D-21266), which was expected to induce less gastrointestinal toxicity. Objectives of the trial were: to determine the maximum-tolerated dose (MTD) for daily administration, to identify the dose-limiting toxicity (DLT) of this schedule, to assess drug accumulation and to determine the relevant pharmacokinetic parameters. 22 patients with advanced solid tumours were treated at doses ranging from 50 to 350 mg/day for 3 weeks, followed by 1 week of rest. Toxicity consisted mainly of gastrointestinal side-effects: nausea was reported by 11 patients (52%, 10 patients Common Toxicity Criteria (CTC) grades 1-2 and 1 patient CTC grade 3), vomiting by 8 (38%, all CTC grades 1-2), and diarrhoea by 9 (43%, 8 patients CTC grades 1-2 and 1 patient CTC grade 3). The severity of these side effects appeared to increase with increasing doses. Another common side-effect was fatigue, occurring in 9 patients (43%). No haematology toxicity was observed. Dose-limiting toxicity (DLT) was not reached, but gastrointestinal complaints led to an early treatment discontinuation in an increasing number of patients at the higher dose levels. Therefore, MTD was established at 200 mg/day. The pharmacokinetic studies suggested dose proportionality.

Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities.

We investigated endocytosis activity, uptake of miltefosine (hexadecylphosphocholine), phospholipid and cholesterol content, the cell cycle, and apoptosis in 13 tumor cell lines (MCF7, MCF7/ADR, KB-3-1, KB-8-5, KB-C1, HeLa, HeLa-MDR1-G185, HeLa-MDR1-V185, CCRF/CEM, CCRF/VCR1000, CCRF/ADR5000, HL-60, HL-60/AR) with different sensitivities to treatment with the antitumor phospholipid analogues miltefosine and D-21266 (octadecyl-(N,N-dimethyl-piperidino-4-yl)-phosphate). In this panel of cell lines, MDR1 (multidrug resistance gene 1)- and MRP1 (multidrug resistance-associated protein)-expressing cells were found to be slightly more resistant to both compounds than sensitive parental cells. No correlation was found between resistance to miltefosine and endocytosis, intracellular concentration of miltefosine, the phospholipid and cholesterol content, induction of apoptosis, or cell cycle alterations in all the cell lines tested. Wild-type p53 containing WMN Burkitt's lymphoma cells and wild type p53-deficient CA46 exhibited similar sensitivities to miltefosine. The low percentage of apoptosis induced in MCF7 cells lacking caspase 3 indicated that caspase 3 seems to play an essential role in miltefosine-induced apoptosis.

Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry.

A HPLC assay with tandem mass spectrometric detection in the positive-ion atmospheric pressure chemical ionisation (APCI) mode for the sensitive determination of retigabine [(I), D-23129] and its acetyl metabolite [(II), ADW 21-360] in plasma was developed, utilising the structural analogue (D-10328), (III), as internal standard. Automated on-line solid-phase extraction of diluted plasma samples, based on 200-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of retigabine and the acetyl metabolite down to 1 ng/ml. Injection of 500 microl of diluted plasma onto a C2 stationary phase-based column switching system in combination with a 75 mm x 4 mm reversed-phase analytical column at a flow-rate of 0.5 ml/min provided cycle times of 4 min per sample. The standard curves were linear from 1 to 1000 ng/ml using weighted linear regression analysis (1/x2). The method is accurate (mean accuracy < or = +/- 10%), precise (RSD < +/- 15%) and sensitive, providing lower limits of quantification in plasma of 1 ng/ml for retigabine (I), and 2.5 ng/ml for the metabolite (II) with limits of detection of 0.5 ng/ml for both analytes. Up to 200 unknowns may be analysed each 24 h per analyst.

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography.

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Rapid and automated determination of the beta2-agonist reproterol in human plasma by atmospheric pressure chemical ionisation high-performance liquid chromatography-tandem mass spectrometry.

An on-line HPLC assay with tandem mass spectrometric detection for the fast and sensitive determination of reproterol in human plasma was developed, utilising a methylated structural analogue as the internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250 microl plasma aliquots, at pH 6.5, allowed a reliable quantification of reproterol down to 400 pg/ml. Injection of 100 microl of plasma extracts onto a 30 mm x 4.6 mm reversed-phase guard column provided retention times ranging from 20 to 30 s for reproterol and the internal standard. The standard curves were linear from 0.2 to 200 ng/ml using weighted linear regression analysis (1/y2). The inter-assay and intra-assay accuracies were -0.9% and +3.2%, exhibiting a precision (C.V.) of +/- 11% and +/- 9.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Rapid quantitative determination of a collagenase inhibitor and its major metabolite by on-line liquid chromatography with ionspray tandem mass spectrometric detection.

A rapid, sensitive and specific analytical method has been developed and validated to quantify the collagenase inhibitor N2-(2(5)-[(hydroxycarbamoyl)methyl)-4-methylvaleryl]-N1, 3-dimethyl-L-valin-amide (I) and its major metabolite (II) from plasma and urine. This method employs an automated solid-phase extraction procedure to isolate the analytes and the internal standard from the biological matrix. Reconstituted extracts were analyzed by HPLC-ionspray MS-MS. Chromatography was performed on a 4.6 mm I.D. reversed-phase guard column. The retention times of the analytes and the internal standard were approximately 1.3 min. The assay has a limit of quantification of 5 ng/ml plasma and a limit of detection of 1 ng/ml, based on 100-micro l plasma aliquots. No sample-drying step was required. The standard curves were linear form 5 to 5000 ng/ml using weighted linear regression analysis (1/y2). The intra- and inter-assay precision were better than + or - 10% with intra- and inter-assay accuracies between 95 and 105%. this new HPLC-MS-MS assay procedure for I and II in plasma and urine has proven to be specific, sensitive, accurate and robust, and is being used routinely for the analysis of I in pre-clinical and clinical trial samples. Up to 200 unknowns may be analyzed each 24 h per analyst.

Estimation of the absolute bioavailability of flecainide using stable isotope technique.

Data on the absolute bioavailability of flecainide are controversial. We have investigated whether differences in metabolic clearances and/or the absorption profile might be responsible for the variability in its absolute bioavailability. Six subjects with a wide range of flecainide metabolic clearances (85-407 ml.min-1) simultaneously received the drug by the IV and oral routes; the oral dose was labelled with deuterium. Besides estimation of absolute bioavailability, this design permitted assessment of metabolic clearance after IV and oral administration, and absorption could be assessed from the urinary excretion of labelled and unlabelled drug and metabolites. The absolute bioavailability of flecainide ranged from 79.9 to 101.1% (mean 93.6%). The absorption was 86.1 to 101.3% (mean 93.2%). The data indicate that the variable bioavailability of flecainide is due both to metabolism and absorption. The study highlights the potential of stable isotope technique in the investigation of such issues.

Variable disposition kinetics and electrocardiographic effects of flecainide during repeated dosing in humans: contribution of genetic factors, dose-dependent clearance, and interaction with amiodarone.

We studied the influence of cytochrome P450 2D6 (CYP2D6) on the steady-state disposition kinetics and the electrocardiographic effects of flecainide at two doses and during combination with amiodarone. Seven extensive and five poor metabolizers of dextromethorphan were studied during a three-period crossover study. All subjects received 50 mg flecainide every 12 hours, alone or together with 200 mg amiodarone every 12 hours, and 100 mg flecainide every 12 hours for 5 days. Mean steady-state plasma concentration of flecainide and QRS change from predrug value did not differ significantly among extensive and poor metabolizer subjects during each study period. Except for a shortened elimination half-life and nonlinear kinetics in extensive metabolizer subjects, phenotype had no significant influence on flecainide pharmacokinetics. Combination with amiodarone resulted in an increase in mean flecainide plasma concentration and effect in subjects with both phenotypes. Our findings indicate that CYP2D6 phenotype predicts flecainide nonlinear kinetics and flecainide half-life but has no influence on electrocardiographic effects during repeated administration of flecainide or on the extent of the amiodarone-flecainide interaction.

Investigations of mechanisms of reactive metabolite formation from (R)-(+)-pulegone.

1. (R)-(+)-Pulegone is a monoterpene that is oxidized by cytochromes P-450 to reactive metabolites that initiate events in the pathogenesis of hepatotoxicity in mice, rats and humans. 2. Selective labelling of (R)-(+)-pulegone with deuterium revealed that menthofuran was a proximate hepatotoxic metabolite formed by oxidation of the allylic methyl groups of pulegone. Incubations of pulegone with mouse liver microsomes in an atmosphere of 18O2 resulted in the formation of menthofuran that contained only oxygen-18 in the furan moiety. These results are consistent with oxidation of pulegone to an allylic alcohol that reacts intramolecularly with the ketone moiety to form a hemiketal that subsequently dehydrates to generate menthofuran. 3. Studies on the metabolism of menthofuran revealed that it is oxidized by cytochromes P-450 to an electrophilic gamma-ketoenal that reacts with nucleophilic groups on proteins to form covalent adducts. In addition, diastereomeric mintlactones are formed. Investigations with H2(18)O and 18O2 are indicative of a furan epoxide intermediate, or a precursor, in the formation of the gamma-ketoenal and mintlactones.

Reactive intermediates in the oxidation of menthofuran by cytochromes P-450.

Menthofuran, a naturally occurring hepatotoxin, is metabolically activated to chemically reactive intermediates that are capable of covalent binding to cellular proteins. Studies in vivo and in vitro with inhibitors and inducers of hepatic cytochromes P-450 demonstrated an association between hepatocellular damage caused by menthofuran and its metabolic activation and covalent binding to target organ proteins. The same gamma-ketoenal formed from the metabolic precursor of menthofuran, pulegone, is the major electrophilic metabolite of menthofuran as well. Diastereomeric mintlactones also are formed, and studies with H218O and 18O2 indicate that the gamma-ketoenal is a precursor to the mintlactones, as well as other reactive intermediates in the cytochrome P-450 mediated oxidation of menthofuran.

[Antitumor platinum(II) complexes with substituted 2-aminomethylpyridine ligands]. / Antitumoraktive Platin (II)-Komplexe mit substituierten 2-Aminomethylpyridin-Liganden.

Reaction of K2PtCl4 with the substituted 2-aminomethylpyridines 9, 14, and 22 affords the corresponding dichloroplatinum(II) complexes 3-5. Compounds 3 and 22 show remarkable relative binding affinities for the estrogen receptor. Towards the hormone-independent P388-tumor of the CD2F1-mouse the platinum(II) complexes 4 and 5 are weakly active, complex 3 is inactive. Towards the hormone-independent MDA-MB 231-cell line, compounds 3-5, 9, 14, and 22 exhibit no significant antitumor activity. Towards the hormone-dependent MCF-7 cell line, compounds 3-5, 9, 14 show weak antitumor activity, whereas compound 22 exhibits strong inhibition.

2-Phenylindole-linked [2-(aminoalkyl)pyridine]dichloroplatinum(II): complexes with a selective action on estrogen receptor positive mammary tumors.

A number of [2-(aminomethyl)pyridine]dichloroplatinum(II) complexes, linked to 5-hydroxy-2-(4-hydroxyphenyl)indoles by alkyl spacer groups of varying lengths, were synthesized and studied for their binding affinities for the calf uterine estrogen receptor. Their relative binding affinity (RBA) values ranged from 1.0 to 5.2% (estradiol, RBA = 100%). Highest affinities were found with complexes possessing a (CH2)5- or (CH2)6-bridge between the pyridine aminomethyl group and the indole nitrogen. Endocrine activities of the complexes and their ligands, determined in the mouse uterine weight test, were low. All compounds entered comparative tests using estrogen receptor positive and negative mammary tumor models. In cell culture, a growth inhibiting effect was only observed in hormone-sensitive MCF-7 cells, but not in hormone-independent MDA-MB 231 cells. In this assay, there was no significant difference between complexes and their ligands. In vivo, the growth of estrogen receptor positive MXT mouse mammary tumors was strongly inhibited by the complexes whereas the hormone-independent MXT mammary tumors showed only a minor response. At a dose of 3 X 20 mg/kg per week, complexes 10d-g reduced the tumor weight by ca. 80% after 6 weeks of treatment. This effect was generally stronger than that exerted by the ligands. The doses applied were well tolerated. Since the complexes described in this paper require the estrogen receptor for their action, a mechanism similar to that of antiestrogens is assumed.

1-(aminoalkyl)-2-phenylindoles as novel pure estrogen antagonists.

A number of 1-(omega-aminoalkyl)-5-hydroxy-2-(4-hydroxyphenyl)indoles were synthesized and studied for their binding affinities for the calf uterine estrogen receptor and estrogen antagonistic activities. Highest binding affinities were found with derivatives bearing a methyl group in position 3 and a hexamethylene chain between the indole and amino nitrogen atoms. Values for relative binding affinity (RBA) are between 20 and 30 for derivatives 5b, 5c, 5f, and 5h (17 beta-estradiol = 100). In the mouse uterine weight test, no significant agonistic (estrogenic) activity was observed up to a daily dose of 125 micrograms/animal, except for derivatives 5c, 5j, and 5l. 2-Phenylindoles with amino (5b), pyrrolidino (5f), piperidino (5h), and morpholino (5k) groups as the amino function completely suppressed estrone-stimulated uterine growth as a dose of 125 micrograms/animal (100% antagonism). Therefore, these derivatives can be considered as first examples of nonsteroidal pure antiestrogens.

Platinum complexes with a selective action on estrogen receptor-positive mammary tumors.

Four (1,3-diaminopropane)dichloroplatinum(II) complexes, linked to 5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole by spacer groups of varying lengths, were synthesized and studied for their binding affinities for the calf uterine estrogen receptor. The RBA-values ranged from 1.0 to 4.4 (estradiol: RBA = 100). The endocrine activities of the complexes and their ligands, determined in the mouse uterine weight test, are low. All compounds entered comparative tests using estrogen receptor-positive and negative mammary tumors models. The receptor levels in these tumors were determined by a modified h.p.l.c. micro assay. In cell culture, a growth inhibiting effect was only observed in hormone-sensitive MCF-7 cells, but not in hormone-independent MDA-MB-231 cells. At 10(-6) molar, the cell number was generally decreased by 50%. In vivo, the growth of estrogen receptor-positive MXT mouse tumors was strongly inhibited whereas the hormone-independent MXT mammary tumors showed only a minor response. The most active compound was the platinum complex with a xylidene spacer group (4d-PtCl2) showing a reduction of tumor weight of 84% after 6 weeks of treatment (3 x 20 mg/kg/week). One of the complexes (4c-PtCl2) and its ligand were tested for activity against the hormone sensitive DMBA-induced rat mammary carcinoma. The inhibitory effect of the complex was close to that of cisplatinum. In these experiments, no sign of toxicity was observed. The selective effect on estrogen receptor-positive tumors make an endocrine mode of action both for the complexes and their ligands likely.

Carrier mediated action of platinum complexes on estrogen receptor positive tumors.

Three 1,2-diaminoethane-dichloro-platinum(II) complexes linked to 5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole by spacer groups of varying length were evaluated for cytostatic activity in estrogen receptor (ER) positive and negative tumor cells. In vitro, only the growth of ER positive MCF-7 mammary tumor cells was inhibited whereas hormone independent MDA-MB 231 cells did not respond. In vivo, a strong inhibitory effect was only observed in ER positive MXT mammary tumors of the mouse. The complex with a hexyl group as spacer reduced the tumor weight by 89% after 6 weeks of treatment. The R 3327 Dunning prostatic tumor of the rat, which also contains ER was inhibited, too. Generally, the effect in ER negative tumors was weak. These findings can be rationalized by the high binding affinities of the complexes for ER. By the mouse uterine weight test it was shown that the endocrine activity of the complexes is very low. Therefore, a mode of action different from that exerted by estrogens or antiestrogens has to be assumed.

Platinum complexes with binding affinity for the estrogen receptor.

A number of (1,2-diaminoethane)dichloroplatinum(II) complexes, linked to dihydroxy-2-phenylindole by spacer groups of varying lengths, were synthesized and studied for their binding affinities for the calf uterine estrogen receptor. Best binding conditions were provided by the n-hexyl and the p-xylene group as spacer with RBA values of 6.5 (16c) and 4.4 (17c), respectively (17 beta-estradiol: RBA = 100). These values are only slightly lower than those of the corresponding diaminoethane ligands.