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Trofosfamide and its congeners ifosfamide and cyclophosphamide are cell-cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P450 (CYP) enzymes. Two NADPH-dependent metabolic routes for the anticancer drug trofosfamide, i.e., 4-hydroxylation and N-dechloroethylation, were studied in human liver microsomes and in seven recombinant human CYP isoforms (i.e., CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4-OR) to identify the CYP enzymes involved. Recombinant human CYP3A4 and CYP2B6 exhibited catalytic activity with respect to both pathways of trofosfamide. Enzyme kinetic analyses revealed the dominant role of human CYP3A4 in 4-hydroxylation and N-dechloroethylation of trofosfamide. This was confirmed by the observation that only the CYP3A4 contents of five samples of human liver microsomes correlated with both pathways of trofosfamide. Furthermore, ketoconazole, a selective inhibitor of CYP3A4, substantially inhibited microsomal trofosfamide 4-hydroxylation and N-dechloroethylation (50% inhibitory concentration < 1 microM for both reactions). The present study indicates that human liver microsomal CYP3A4 preferentially catalyzes the two NADPH- dependent metabolic routes of trofosfamide, which emphasizes the necessity for awareness of potential interactions with any coadministered drugs that are CYP3A4 substrates. [ABSTRACT FROM AUTHOR]

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.

Background: Intravenous pulse administration of cyclophosphamide (CYC) has been successfully used for the treatment of various autoimmune diseases. These patients often present with impaired renal function or even end-stage renal failure. Nevertheless, data concerning pharmacokinetics of CYC in renal insufficiency (RI) and on hemodialysis (HD) are rare and contradictory., Methods: The pharmacokinetics of CYC (0.5 to 1 g/m2 as a one-hour infusion) were determined in patients with renal involvement of autoimmune diseases. Group A (N = 6) patients had a creatinine clearance (CCr) of 25 to 50 mL/min, group B patients' (N = 5) CCr was 10 to 24 mL/min, and group C (N = 6) patients had CCr values <10 mL/min and HD. Concentrations of CYC in serum, dialysate and urine were measured by HPLC. Twelve previously investigated patients with normal renal function served as controls., Results: Mean clearance (CL) of CYC was significantly reduced with decreased renal function (79 vs. 57 and 47 mL/min, controls vs. A and B, respectively, P < 0.05), but only moderately lower in the patients who received a three-hour HD during the study period (group C, 64 mL/min, NS). This resulted in reciprocal increases in systemic drug exposure (dose corrected AUC was 216, 298, 382 and 266 microg x h/mL x g, controls, A, B and C, respectively). Urinary excretion of CYC was markedly reduced in all patients with RI (renal CL was 14.9 vs. 3.4, 2.4 and 2.1 mL/min, controls vs. A, B and C, respectively, P < 0.001). However, in patient group C, a mean of 22% of administered CYC dose was eliminated by a three hour HD starting seven hours after CYC administration. Individual CCr values were significantly (P < 0.001) correlated with renal and systemic CL of CYC, respectively, and negatively correlated with dose corrected AUC., Conclusions: Clearance of CYC is decreased in patients with reduced renal function, thereby resulting in an increased systemic drug exposure. However, in hemodialysis-dependent patients, removal of CYC into the dialysate has to be taken into account. For optimal dosing of CYC in patients with renal insufficiency, the severity of renal impairment and the use and timing of hemodialysis have to be considered.

Organic-soluble extracts of the twigs of Aglaia oligophylla collected in Vietnam yielded four insecticidal cyclopentatetrahydrobenzofurans of the rocaglamide type including one new natural product (compound 4). Moreover, two cyclopentatetrahydrobenzopyran derivatives, belonging to the aglain and aglaforbesin types, respectively, were also isolated. The aglaforbesin derivative 6 proved likewise to be a new natural product. All isolated rocaglamide, aglain, and aglaforbesin derivatives have a characteristic methylenedioxy substituent linked to C-6 and C-7 or to C-7 and C-8, respectively. Structure elucidation of the new natural products and the determination of the absolute configuration of compound 1 by calculation of its CD spectrum with molecular dynamics simulation are described. All isolated rocaglamide derivatives exhibited strong insecticidal activity toward neonate larvae of the polyphageous pest insect Spodoptera littoralis when incorporated into an artificial diet, with LC(50) values varying between 2.15 and 6.52 ppm.

Background: Today knowledge about pharmacokinetics of anticancer drugs in human malignant tumors is poor. Data from in vivo studies are limited and difficult to obtain due to ethical aspects. An ex vivo isolated perfused and ventilated human lung model however allows pharmacological studies of human bronchial carcinoma inside their host organ, the lung, under physiological conditions without compromising the patient., Methods: Following surgery for bronchial carcinoma human lung preparations were reperfused and ventilated extracorporally for 2-3 hours. During the reperfusion anticancer drugs are added to the perfusion solution and their uptake into tumor, normal lung tissue, and lymph nodes is studied., Results: An initial study showed that lung reperfusion under physiological circumstances over a period of 2-3 hours did not interfere with histo-pathological diagnostics and staging; an important precondition for potential adjuvant treatment. Pharmacokinetics of cyclophosphamid, adriamycin, and a water soluble adriamycin prodrug (HMR 1826) were measured. Final tissue concentrations of adriamycin and cyclophosphamid in peripheral lung parenchyma turned out to be 10 times higher compared to tumor tissue. However, following perfusion with adriamycin prodrug final tissue concentrations of adriamycin were in the same range in lung and tumor., Conclusions: The ex-vivo isolated human lung perfusion model (IHLP) has proven to be an ideal scientific model for pharmacological investigations of human tumors as an intermediate step between cell culture and in-vivo situation without any disadvantage for the patient. The tumor-to-host interaction is completely saved in this model. However, first pass reactions of drugs in other organs must not play a role for the substances studied with the IHLP. The role and future applications of the isolated perfused human lung model for other indications is discussed.

Purpose: The alkylating cytostatic prodrug cyclophosphamide is bioactivated by the human cytochrome P450 enzyme system. Since these enzymes are not only expressed in human liver, but also in extrahepatic tissue, local bioactivation of this drug may play an important role in its antineoplastic effects, e.g., chemotherapy of lung tumors. This would require uptake of significant amounts of cyclophosphamide into tumor tissue, which has not yet been demonstrated., Methods: We used a recently developed, ex vivo isolated, ventilated and perfused human lung model to study cyclophosphamide uptake into bronchial carcinoma and healthy lung tissue. Following a standard lobectomy, lung samples containing the tumor were perfused with buffer containing 2 mM cyclophosphamide for 2 h. Cyclophosphamide concentrations in perfusate and healthy peripheral tissue were measured during the perfusion and in tumors at the end of perfusion., Results: In all tissue samples, cyclophosphamide uptake was relatively poor, indicated by a tissue to perfusate ratio of 0.021. Moreover, in tumor samples, cyclophosphamide concentrations were significantly lower (P < 0.05) than in healthy lung tissue and showed pronounced interindividual variability. Median concentrations were 36.8 microg/g (26.9 44.2 microg/g) in healthy tissue and 5.1 microg/g (0.0-26.8 microg/g) in tumor samples. Tumor cyclophosphamide concentrations varied between 0 and 75% of those reached in healthy tissue., Conclusions: Our results indicate that CP tumor concentrations are modulated by factors different from dose and that expression of bioactivating enzymes in human lung or transfection of genes encoding these enzymes into tumor cells does not necessarily lead to local bioactivation of cyclophosphamide.

Thirteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener all of them isolated from three Aglaia species (Aglaia duperreana, A. oligophylla and A. spectabilis) collected in Vietnam were studied for their antiproliferative effects using the human monocytic leukemia cell lines MONO-MAC-1 and MONO-MAC-6. Only rocaglamide type compounds showed significant inhibition of [3H-]thymidine incorporation and the most active compound didesmethylrocaglamide inhibited cell growth in a similar concentration range as the well-known anticancer drug vinblastine sulfate. Detailed structure-activity analysis indicated that the OH-group at C-8b which is a common structural feature of most naturally occurring rocaglamide compounds is essential for the described antiproliferative activity since replacement of this group by methylation led to a complete loss of the inhibitory activity for the resulting derivative. Rocaglamide derivatives rapidly inhibited DNA as well as protein biosynthesis of MONO-MAC-6 cells at concentrations well below those of actinomycin D or cycloheximide which were used as positive controls in the respective experiments. Didesmethylrocaglamide was furthermore able to induce growth arrest of MONO-MAC-1 cells in the G2/M and probably G0/G1-phase of the cell cycle with no morphological indication of cellular damage. Our data suggests that 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type act primarily by a cytostatic mechanism.

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human beta-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 microM (38 ng/ml) and a limit of quantitation of 1 microM (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human beta-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human beta-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.

Purpose: The alkylating agent cyclophosphamide (CP) is a prodrug that is metabolized to both cytotoxic and inactive compounds. We have previously shown that following dose escalation from conventional-dose (CD) to high-dose (HD) levels; the fraction of the dose cleared by bioactivation is significantly decreased (66% versus 48.5%) in favor of inactivating elimination pathways when the HD is given as a single 1-h infusion. Based on the concept of bioactivating enzyme saturation with increasing doses, we investigated the influence of fractionated application of HD-CP on dose-dependent changes in metabolism., Patients and Methods: Plasma concentrations of CP (measured by high-performance liquid chromatography, HPLC) and urinary concentrations of CP and its major metabolites (quantified by [31P]-nuclear magnetic resonance spectroscopy; [31P]-NMR spectroscopy), were determined in four patients with high-risk primary breast cancer who received adjuvant chemotherapy including both CD-CP (500 mg/ m2 infused over 1 h) and split HD-CP (50 mg/kg infused over 1 h on each of 2 consecutive days (d): d1 and d2., Results: (Data are given as mean values for CD and d1/d2 of HD, respectively). Systemic clearance (CL) of CP was similar during CD and d1 of HD, but significantly increased on d2 of HD (CL: 83 and 78/115 ml/min; P < 0.01 for d1 versus d2). The latter was translated into an increase in formation CL of both active (+ 16.4 ml/min) and inactive metabolites (+ 17.6 ml/ min) and reflects autoinduction of metabolism. As compared with CD-CP, no statistically significant decrease was observed in the relative contribution of bioactivation CL to overall CL during both days of HD (63% versus 57%/53%). Recovery of intact CP in 24-h urine corresponded to 24%, 29%, 22% of the dose (P < 0.05 for d1 versus d2 of HD)., Conclusions: Following dose escalation of CP, dividing the high dose over 2 days instead of one single infusion may favorably impact the metabolism of CP in terms of bioactivation. In addition, on day 2 of a split regimen, renal elimination of CP is decreased, which implies that more drug is available for metabolism.

Eleven rocaglamide derivatives (cyclopentatetrahydrobenzofurans) and one structurally related aglain congener all isolated from different Aglaia species (Meliaceae) were tested for growth inhibiting properties using the human cancer cell lines MONO-MAC-6 and MEL-JUSO. Proliferation of both cell lines was efficiently inhibited in a dose and compound dependent manner. Applying MTT-Assay, the IC50 of the most active compound didesmethyl-rocaglamide (1) was observed at 0.002 and 0.006 micrograms/ml (0.004 and 0.013 microM) depending on the cell line investigated. Bulky aminoacyl substituents at C-2, acetylation of the OH substituent at C-1 or insertion of a OH or OMe substituent at C-3 of the rocaglamide skeleton all diminished the activity of the compounds investigated. The aglain derivative 12 was inactive up to a concentration of 3 micrograms/ml (4.6 microM). This loss of activity is assumed to be mainly due to the presence of a pyran ring in the aglains vs. a furan ring as found in rocaglamide derivatives. Rocaglamide derivatives may act primarily by inhibition of cell proliferation as evidenced by the absence of a significant cytotoxic effect in long-term cultures of MONO-MAC-6 cells treated with high doses of didesmethylrocaglamide. Our data suggest that rocaglamide derivatives could exert a potential role in the treatment of malignant diseases and are worth to be investigated in further studies of experimental medicine and pharmacology.